Serum resistance factors of gram positive bacteria

ABSTRACT

A newly identified serum resistance factor of gram positive bacteria can be used to treat or prevent bacterial infection.

This application is a division of Ser. No. 11/920,274 filed on Nov. 13,2007 as a national phase application of PCT/US2006/018411 filed May 12,2006, which claims the benefit of and incorporates by referenceco-pending provisional application Ser. No. 60/680,479 filed May 13,2005 and Ser. No. 60/740,291 filed Nov. 29, 2005.

This application incorporates by reference the contents of a 106 kb textfile created on Dec. 15, 2011 and named “PAT051803_sequencelisting.txt,”which is the sequence listing for this application.

FIELD OF THE INVENTION

This invention is in the fields of immunology and vaccinology. Inparticular, it relates to a newly identified serum resistance factor ofgram positive bacteria and its use in compositions for the treatment andprevention of bacterial infection.

BACKGROUND OF THE INVENTION

The gram positive bacteria Group B Streptococcus (GBS) is one of themost important causes of life-threatening bacterial infection in newborninfants, pregnant women, the elderly and individuals with chronicillness. Other gram positive bacteria such as Streptococcus pyogenes(GBS), Streptococcus pneumoniae (Strep pneumo), and Staphylococcusaureus (Staph) are also implicated in significant morbidity andmortality worldwide.

Various streptococci express on their surface multifunctional proteinsthat mediate both bacterial adhesion and acquisition of immune systemcomponents, contributing to a successful colonization of host mucosalsurfaces (Jarva et al., 2003; Talay, 2005). In particular, Streptococcusagalactiae (GBS) and Streptococcus pyogenes (GAS) express a number offunctionally-related proteins, characterized by their capacity to bindboth human immunoglobulins (Boyle, 1998) and fluid-phase complementregulators (Jarva et al., 2004; Lindahl et al., 2005). In GBS, receptorsfor IgA and/or IgG belong to the M protein family (Stenberg et al.,1992); M proteins interact with the type II Fc region of immunoglobulinsoutside their antigen-combining site (Cunningham, 2000).

In GBS, the Bac protein (beta antigen) binds with high affinity to theFc part of human serum IgA (Bevanger, 1983; Johnson and Ferrieri, 1984;Lindahl et al., 1990; Russell-Jones et al., 1984) and to complementregulator Factor H (FH), which avoid C3b deposition on GBS surface(Areschoug et al., 2002). The binding site for IgA has been located tothe N-terminal half of the protein, while the FH-binding region is atthe C-terminal half of Bac (Areschoug et al., 2002; Jarva et al., 2002).Bac is structurally related to the pneumococcal Hic protein, and theybind FH in an analogous fashion (Janulczyk et al., 2000; Jarva et al.,2004).

On the other hand, GAS acquires FH by M proteins and Fba, whichcontributes to the bacterium's capacity to evade phagocytosis bypolymorphonuclear cells (Horstmann et al., 1988; Pandiripally et al.,2002; Pandiripally et al., 2003). M-proteins also mediate acquisition ofC4 binding protein (C4bp), an important regulator of complementclassical pathway component C3 convertase (C4b2a) (Berggard et al.,2001; Blom et al., 2004). M protein binding to C4b has both decayaccelerating activity and cofactor activity for C4b cleavage in ananalogous fashion as FH in the alternative pathway (Carlsson et al.,2005; Perez-Caballero et al., 2004; Thern et al., 1995).

GAS and GBS also secrete the C5a peptidase, a multifunctional enzymethat inactivates human C5a (Jarva et al., 2003; Wexler et al., 1985) andbinds fibronectin, which promotes bacterial invasion of epithelial cells(Beckmann et al., 2002; Cheng et al., 2002).

Serum resistance factors are thought to play a role in mechanisms thesegram positive bacteria use to evade the host immune response. There is,therefore, a continuing need in the art for identification of novelserum resistance factors in gram positive bacteria which can be used todevelop compositions for the prevention or treatment of bacterialinfection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Diagram of BibA inserted in the cell membrane.

FIG. 1B, Comparison of BibA and M protein structures.

FIG. 2A. Domains of BibA proteins in different GBS strains.

FIG. 2B. Representation of BibA cloned fragments, based on predictedfunctional domains.

FIGS. 3A-B. Results of experiments demonstrating that recombinant BibAprotein forms dimers. FIG. 3A, Gel-filtration of recombinant BibA; FIG.3B, Coomassie staining.

FIGS. 4A-B. BibA protein surface-association in GBS strains. FIG. 4A,strains 2603, 18RS21, and H36B. FIG. 4B, strains 515 and CJB111.

FIGS. 5A-B. Data demonstrating that BibA protein is associated with thebacterial cell membrane or in the supernatant of GBS cultures. FIG. 5A,FACS analysis and Western blot (strains 2603 type V and 18RS21 type II;FIG. 5B, FACS analysis and Western blot (strain H36B type 1b).

FIG. 6. Micrographs showing immunogold staining of strain 515, whichexpresses BibA protein of the 2603 V/R strain.

FIG. 7A. Drawing showing BibA gene from strain 2063 cloned into pAM401vector.

FIG. 7B. FACS analysis demonstrating that BibA is expressed on thesurface of strain 515 pAM401.

FIG. 8A. FACS analysis demonstrating increased BibA expression on thesurface of strain 2603.

FIG. 8B, FACS analysis demonstrating BibA expression on the surface ofstrain 515.

FIG. 9. FACS analysis demonstrating human-IgA-FITC binding to thesurface of 2603-BibA overexpressing mutant strain.

FIGS. 10A-E. Data demonstrating portions of BibA associated with thebacterial cell membrane or in the supernatant of GBS cultures. FIG. 10A,strain 515 type 1a; FIG. 10B, strain CJB111 type V; FIG. 10C, strain2603 V/R; FIG. 10D, strain 18RS21; FIG. 10E, strain H36B.

FIGS. 11A-B. Blots showing that BibA binds to C4 binding protein (C4BP).FIG. 11A, dot blot of native GBS proteins and GBS M1 protein overlaidwith C4BP and probed with anti-C4BP antibody; FIG. 11B, Western blotanalysis of recombinant GBS proteins overlaid with C4BP and probed withanti-C4BP antibody (left, Ponceau staining).

FIG. 12. FACS analysis of BibA binding to the surface of variousepithelial cells.

FIG. 13. FACS analysis of BibA fragment binding to epithelial cells.

FIGS. 14A-C. Western blots of BibA protein overlaid with purified humanIgG and probed with anti-human IgG-HRP conjugated antibody. FIG. 14A,7.5 pMol of each protein overlaid with 5 mg/ml purified human IgG; FIG.14B, 7.5 pMol of each protein overlaid with 1 mg/ml purified human IgG;FIG. 14C, 15 pMol=BibA overlaid with 5 μg/ml purified human IgG.

FIG. 15. Blots showing that purified human-IgA binds to BibA protein.Left two blots, Western blot analysis of denatured BibA overlaid withpurified human IgA-HRP. Right blot, dot blot analysis of native BibAoverlaid with purified human IgA-HRP.

FIG. 16. Western blot showing binding of tryptic digested fragments ofBibA to IgA.

FIGS. 17A-C. Blots demonstrating that BibA-His is specific for human andrabbit IgG. FIG. 17A, human serum goat-α-human-IgG-HRP; FIG. 17B, rabbitserum goat-α-rabbit-IgG-HRP; FIG. 17C, mouse serumrabbit-α-mouse-IgG-HRP.

FIG. 18. Western blot showing that BibA binds to human IgG (lane C),human serum IgA (lane E) and C4BP (lane G). M protein of GBS was used aspositive control (lanes D, F, H).

FIGS. 19A-L. Data demonstrating that BibA is expressed as surfaceexposed and secreted in GBS strain 2603 V/R. FIG. 19A, flow cytometryanalysis of BibA on the surface of 2603 V/R GBS strain. Bacteria wereincubated with a polyclonal mouse anti-BibA antibody and stained withFITC-conjugated anti-mouse IgG antibody black line histogram. The dashedline histogram indicates bacteria treated with primary and secondaryantibodies alone.

FIG. 19B, immunogold electron microscopy of BibA expression on GBSstrains 2603 V/R. Bacteria were absorbed to formvar-carbon-coated nickelgrids and then fixed in 2% PFA. The grids were floated on drops ofprimary antiserum anti-BibA protein and then on secondary antibodyconjugated to 10-nm gold particles.

FIG. 19C, Western blot analysis of the presence of BibA in proteinextracts from GBS strain 2603 V/R. P Peptidoglycan associated proteinfraction and S Bacterial supernatant protein fraction. GBS proteinfractions were separated on SDS-10% PAGE gels and transferred tonitrocellulose membrane. Proteins were overlaid with a mouse anti-BibApolyclonal antibody and stained with HRP-conjugated antibody. Positivebands were detected by ECL.

FIG. 19D, flow cytometry analysis of BibA on the surface of strain2603ΔbibA. Bacteria were incubated with a polyclonal mouse anti-BibAantibody and stained with FITC-conjugated anti-mouse IgG antibody blackline histogram. The dashed line histogram indicates bacteria treatedwith primary and secondary antibodies alone.

FIG. 19E, immunogold electron microscopy of BibA expression on GBSstrains 2603ΔbibA. Bacteria were absorbed to formvar-carbon-coatednickel grids and then fixed in 2% PFA. The grids were floated on dropsof primary antiserum anti-BibA protein and then on secondary antibodyconjugated to 10-nm gold particles.

FIG. 19F, flow cytometry analysis of BibA on the surface of strain 515Ia. Bacteria were incubated with a polyclonal mouse anti-BibA antibodyand stained with FITC-conjugated anti-mouse IgG antibody black linehistogram. The dashed line histogram indicates bacteria treated withprimary and secondary antibodies alone.

FIG. 19G, immunogold electron microscopy of BibA expression on GBSstrains 515 Ia. Bacteria were absorbed to formvar-carbon-coated nickelgrids and then fixed in 2% PFA. The grids were floated on drops ofprimary antiserum anti-BibA protein and then on secondary antibodyconjugated to 10-nm gold particles.

FIG. 19H, Western blot analysis of the presence of BibA in proteinextracts from GBS strains 515 Ia and 515pAM401bibA. P Peptidoglycanassociated protein fraction and S Bacterial supernatant proteinfraction. GBS protein fractions were separated on SDS-10% PAGE gels andtransferred to nitrocellulose membrane. Proteins were overlaid with amouse anti-BibA polyclonal antibody and stained with HRP-conjugatedantibody. Positive bands were detected by ECL.

FIG. 19I, flow cytometry analysis of BibA on the surface of strain515pAM401bibA. Bacteria were incubated with a polyclonal mouse anti-BibAantibody and stained with FITC-conjugated anti-mouse IgG antibody blackline histogram. The dashed line histogram indicates bacteria treatedwith primary and secondary antibodies alone.

FIG. 19L, immunogold electron microscopy of BibA expression on GBSstrains 515pAM401bibA. Bacteria were absorbed to formvar-carbon-coatednickel grids and then fixed in 2% PFA. The grids were floated on dropsof primary antiserum anti-BibA protein and then on secondary antibodyconjugated to 10-nm gold particles.

FIGS. 20A-F. Western blots demonstrating that BibA binds to humanimmunoglobulins. FIG. 20A, recombinant BibA separated on SDS PAGE andblotted on nitrocellulose membrane. The membrane was then overlaid with0.5 μg/ml human, mouse or bovine purified serum IgG and positive bindingto IgG revealed by secondary antibodies versus the different IgGspecies. To evaluate the binding ECL detection was performed. M1 proteinof GBS was used as positive control, while GBS104 was used as anon-specific binding control. FIG. 20B as in FIG. 20A apart from testingthe binding to human serum or secretory IgA, overlaid at a concentrationof 0.5 μg/ml. The blots are representative of experiments performed atleast in triplicate. FIG. 20C and FIG. 20D Different concentrations ofpurified recombinant BibA in PBS were spotted on a nitrocellulosemembrane and overlay assay performed as in FIG. 20A. FIG. 20C, overlaywith human serum IgG. FIG. 20D, overlay with human serum IgA. FIG. 20E,overlay blotting with human IgG of SDS-PAGE separated N-terminal andC-terminal constructs of BibA. FIG. 20F, overlay blotting with human IgAof SDS-PAGE separated N-terminal and C-terminal constructs of BibA.

FIGS. 21A-C. Data demonstrating that BibA binds to human C4BP. FIG. 21A,recombinant BibA separated on SDS PAGE and blotted on nitrocellulosemembrane. The membrane was overlaid with 5 μg/ml human C4BP and bindingrevealed by secondary antibodies versus C4BP. M1 protein of GBS was usedas positive control, while GBS201 as non-specific binding control. FIG.21B, dot blot of different concentrations of native recombinant BibAspotted on nitrocellulose membrane and overlaid with 5 μg/ml C4BP as inFIG. 21A. FIG. 21C, Western blot of SDS-PAGE separated N-terminal andC-terminal constructs of BibA overlaid with human C4BP. Experimentalblotting conditions as in FIG. 21A.

FIGS. 22A-C. Graphs demonstrating binding of recombinant BibA toepithelial cells. FIG. 22A, ME180 cells were incubated for 1 h at 4° C.with increasing concentrations of recombinant BibA range 0.01-62.5μg/ml. Then cells were washed and incubated with mouse anti-BibAantibodies followed by FITC-conjugated secondary anti-mouse antibodies.MFI Mean fluorescence intensity. The plot is representative of threeindependent experiments. FIG. 22B, saturation curve of BibA binding toME180 cells. Analysis was performed on data reported on panel A. The Kdvalue was calculated as the BibA concentration that determines thesaturation of 50% of the receptors present on cells. FIG. 22C,representative flow cytometric profiles of the binding of 10 μg/ml BibAto A549, Caco2 and 16HBE epithelial cells. Binding experimentalconditions and analysis as in FIG. 22A. Dashed-line histograms representthe MFI of control cells.

FIGS. 23A-F. Data demonstrating that BibA expression modulates GBScapacity to adhere to epithelial cells. FIG. 23A, ME180 cells grown in a24 well plate were infected with GBS strains 2603 V/R, 2603ΔbibA and2603pAM401bibA for 3 hours. Non-adherent bacteria were gently washed offand cells lysed with saponin for association assay. The white columnindicates the percentage of associated bacteria in the wild type strain,the light grey column indicates the percentage of association of theBibA isogenic mutant strain and the dark grey column the association ofthe wild type strain overexpressing BibA. FIG. 23B, as in FIG. 23Aexcept that infection was carried out in A549 cells. Meanvalues±standard deviations of three individual experiments. Dataevaluated by Student's T-test, were 95% confident.

FIG. 23C, micrographs of confocal imaging analysis of the 2603 V/Rstrain association to A549 lung epithelial cells in comparison to theisogenic mutant strain lacking BibA gene (FIG. 23D). A549 cells weregrown on glass slides were infected with GBS for 3 h. Bacteria were thenstained with mouse polyclonal antisera raised against type V capsularpolysaccharide and rabbit polyclonal anti-BibA antibodies. Capsule andBibA were respectively labeled with Alexa Fluor 562 red and 488 greenconjugated secondary antibody. A549 cells F-actin was labeled with AlexaFluor 622 conjugated phalloidin blue. The results shown in the figureare typical of multiple experiments.

FIG. 23E, confocal imaging analysis of the 515 Ia wild type strain andthe isogenic strain carrying a plasmid containing the 515 pAM401bibAgene (FIG. 23F) in association to A549 epithelial cells.

FIG. 24. Overview of sequence organization of BibA proteins. N-terminaldomains are predicted to form helix-rich structures, according toprediction obtained using the Paircoil program Berger et al., 1995 atExpasy web server (domain name expasy.org). Positions of classical LPXTG(SEQ ID NO:3) cell wall anchoring motif and trans-membrane domain arealso indicated.

FIG. 25. Summary of data demonstrating that BibA is involved in GBSadherence to epithelial cells.

FIG. 26. Summary of data demonstrating that BibA overexpressionincreases GBS adherence to epithelial cells.

FIG. 27. Summary of data demonstrating that expression of BibA on 515 Iasurface increases adherence to epithelial cells.

FIG. 28. Summary of data demonstrating that IgA binds to the N-terminalportion of recombinant BibA.

FIG. 29. Summary of data demonstrating that the IgA binding domain iscontained within the first 200 amino acids of BibA.

FIG. 30. BibA promotes GBS survival of PMN killing. Human neutrophilswere incubated for 3 hours with GBS 2603 V/R and 2603ΔbibA mutantstrains (MOI 1:1) in the presence of human serum (white bars) orcomplement inactivated human serum (grey bars). Percentage of viablebacteria after incubation with neutrophils is reported. A typicalexperiment performed in triplicate is shown. The experiment was repeatedat least three times with similar results.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have identified a serum resistance factor in a gram positivebacteria (GBS) which interacts with host cell complement pathways and isthought to be involved in the invading bacteria's complement resistanceor evasion mechanisms. This newly identified serum resistance factor isreferred to herein as group B streptococcus immunoglobulin-bindingadhesion (BibA) (also known as GBS 3). BibA is a widely expressedprotein, present in 81% of the 31 strains of GBS analyzed (Table 3).

A BLAST search against the non-redundant GenBank database revealed a lowsimilarity of the BibA N-terminal region with a series of gram positiveimmunoglobulin-binding proteins such as the M-protein family of S.pyogenes (22% identity), Bac of S. agalactiae (20% identity), PspC of S.pneumoniae (20% identity), and Mig of S. dysagalactiae (27% identity).BibA shares some similarities with resistance factors of othergram-positive bacteria, such as the Hic-like proteins of S. pneumoniae.

The bibA gene is located between secE and nusG genes. SecE and nusG areco-transcribed in E. coli (Downing et al., 1990) and are adjacent in alarge panel of gram positive and gram negative bacteria (Barreiro etal., 2001; Fuller et al., 1999; Jeong et al., 1993; Katayama et al.,1996; Miyake et al., 1994; Poplawski et al., 2000; Puttikhunt et al.,1995; Sharp, 1994; Syvanen et al., 1996). This evidence suggests thatthe present genomic localization of BibA is likely to derive from aninsertion event. Of interest, two transposases present in A909 strainare members of the IS1381 family, which has been proposed as a tool forGBS subtyping (Tamura et al., 2000) and whose presence has beencorrelated with the evolution of the S. agalactiae species analyzed bymultilocus sequence typing (MLST) (Hery-Arnaud et al., 2005).

In silico analysis of the seven GBS completed genomes revealed that BibAis a modular protein; its sequence variability is mainly due to adifferent number of short amino acid repeats either in N-terminal or theC-terminal domains. Full-length BibA comprises an N-terminal helix-richregion, a C-terminal proline-rich region, a LPXTG (SEQ ID NO:3) motifthat anchors the protein to the cell wall peptidoglycan, and atransmembrane domain. FIGS. 1A, 2B. The coiled-coil domain of BibA iswell conserved across multiple serotypes of S. agalactiae.

BibA is structurally related to the family of M-like proteins of S.pyogenes (GBS) (FIG. 1B). The secondary structure of M proteins isprimarily an α-helical coiled coil structure which forms stable dimers(Phillips et al., 1981). In silico prediction of BibA secondarystructure (Berger et al., 1995) reveals in the N-terminal region ahelix-rich region with the propensity to form a coiled-coil arrangement(regions 283-294 and 366-400). Studies on the recombinant form of BibAsuggest that, as for M proteins, BibA is able to form dimers, which areopened in non-reducing conditions. No canonical elements of secondarystructure are on the contrary predicted within the proline-rich region,which suggests that this part of the molecule could adopt a poly-prolinehelix-like conformation.

BibA is expressed on the surface of several GBS strains, but is alsorecovered in GBS culture supernatants. BibA, whether expressed on thecell wall or secreted in the supernatant fractions, has an identicalapparent molecular weight. This suggests that secretion of BibA might bedue to either a proteolytic cleavage of the cell-wall anchoring domainor that an impaired sorting of the protein could be responsible for thesecretion. Indeed, BibA has an YSSIRK-G/S-like motif (SEQ ID NO:64) inthe signal peptide, which has previously been described to be present inStaphylococcus aureus and other gram positive pathogens (Bae andSchneewind, 2003). Such a motif is exclusively present in BibA and isconserved in all the eight GBS strains analyzed. The YSSIRK-G/S-likemotif (SEQ ID NO:64) has been postulated to be involved in acceleratingprotein maturation (Bae and Schneewind, 2003). Based on this evidence,we hypothesize that sortase components may be limiting for complete andefficient anchoring of BibA, which results in the incomplete processingof mature BibA and the release of the protein in the supernatant.

Functional characterization identifies BibA as a member of a group ofstreptococcal surface-exposed multifunctional proteins which mediatebacterial colonization and modulate host immune-response (Jarva et al.,2003; Lindahl et al., 2005). However, BibA has unique features, such asthe binding both to human immunoglobulins and to complement regulatorC4bp. The BibA binding site for IgA and C4bp resides in the N-terminalregion of the protein. However, there is no homology to the BacN-terminal domain specific for IgA (Lindahl et al., 1990). The lack ofbinding to mouse and bovine IgG suggests that BibA has a human specificfunctional role, as reported for other Ig-binding proteins.

Secreted BibA binds to human epithelial cells, complement (such as C4binding protein), and specifically to human IgG and IgA. Theproline-rich domain of secreted BibA is responsible for the bindinginteraction with human epithelial cells. Examples 8, 9. The proline-richdomain has a periodicity of 8 amino acids. Proline occupies positions band f of the motif, which is repeated 19 times:

399-(aKPDVKPEAh) (SEQ ID NO: 9)      (KPEAKPDV)6 (SEQ ID NO: 10)      KPKAKPDV (SEQ ID NO: 11)       KPEAKPDV (SEQ ID NO: 10)      KPDVKPDV (SEQ ID NO: 12)       KPEAKPED (SEQ ID NO: 13)      KPDVKPDV (SEQ ID NO: 12)       KPEAKPDV (SEQ ID NO: 10)     (KPEAKPEA)3 (SEQ ID NO: 14)      (KPDVKPEA)2 (SEQ ID NO: 15)      KPEAKPEA-551 (SEQ ID NO: 14)

The proline-rich domain, when present, is located towards the C-terminusof BibA. As illustrated in FIG. 2A, the proline-rich domain is generallylocated from amino acid 400 to the end of the C-terminus.

To elucidate the BibA binding region to immunoglobulins, we generatedtwo constructs comprising the N-terminal or the C-terminal portion ofthe protein. BibA binding to human IgG resides predominantly in theN-terminal region of the protein, while the C-terminal region binds to alower extent. On the other hand, the binding to human IgA wasexclusively associated to the N-terminal portion of BibA. This regionwas also responsible for the binding of BibA to the C4bp. In addition,recombinant BibA binds to human epithelial cells of different origin,with an affinity constant of ˜10⁻⁸ M.

The coiled-coil domain is well-conserved in various GBS strains. Thecoiled-coil domain is responsible for the binding interaction of BibAwith complement such as C4 binding protein. Example 7. The coiled-coildomain is also responsible for binding interactions with humanimmunoglobulins, such as IgG and IgA. Examples 10, 11, and 12. The IgAbinding site appears to be in the N-terminal portion (roughly 200 aminoacids) of BibA. BibA, like other coiled-coil proteins, forms dimers.Example 1.

When bacteria secrete BibA, it is believed that the proline-rich Cterminus domain of the protein binds to host epithelial cells, leavingthe N-terminal coiled-coil domain exposed to serum factors. TheN-terminal coiled-coil domain is then thought to attract complement,such as C4 binding protein, diverting it away from the invadingbacteria. Complement binding interaction with the host cell attachedcoiled-coil domain attracts complement activity to the host cell,further facilitating bacterial invasion.

In some strains the LPXTG (SEQ ID NO:3)/proline-rich domain is absent.FIG. 2A. When the proline-rich domain is absent or expressed separately,BibA is thought to be primarily secreted, and not surface exposed.However, even truncated or bifurcated forms of BibA are thought todivert immune system attention away from the bacterium as it approachestarget host cells.

The role of BibA in GBS adhesion to cells was confirmed by the impairedability of a BibA knock-out mutant strain to bind to both human cervicaland lung epithelial cells. Complementation of the mutation restored GBSadhesive phenotype, while BibA over-expression significantly increasedthe binding to epithelial cells. These characteristics indicate thatBibA is a novel multifunctional protein and is likely involved in GBSpathogenicity.

The soluble form of BibA protein has an apparent molecular weight on anSDS polyacrylamide gel of ˜80 kDa, although its expected molecularweight is ˜60 kDa. The proline-rich domain of the protein is likely tobe responsible for this shift, due to the folding of BibA into abundled-like shape. The membrane-associated form is easily degraded; asmall fraction of the protein runs on a gel as an 80 kD, while the majorfraction runs at a MW of ˜60 kDa. This indicates that in themembrane-associated form the proline-rich motif is still associated withthe cell wall components and maintains a linear structure. See Examples2-4, 6.

Bacterial adherence to host cells is the initial step and a prerequisitefor successful colonization of host mucosal surfaces. The analysis ofthe binding of recombinant BibA to epithelial cells revealed that theassociation to cells could be saturated, with an estimated affinityconstant of ≈4×10⁻⁸ M. In particular, BibA binding to epithelial celllines of lung, intestine, bronchus and cervix origin, suggests theexistence of an ubiquitous receptor. BibA, like M-proteins (Courtney etal., 1994; Courtney et al., 1997; Wang and Stinson, 1994), mediatesbacterial adhesion to epithelial cells. Studies of isogenicBibA-positive and BibA-negative strains indicated that the BibA-positivestrain adhered to epithelial cells, while the BibA-negative strainshowed greatly reduced adherence. In addition, expression of thecell-wall anchored form of BibA in a strain not exposing BibA on thesurface increased its associative phenotype. Of interest, such resultswere confirmed in both human cervical (ME180) and lung (A549) epithelialcell lines, which are a target for GBS colonization.

These functional properties suggest that BibA is a serum resistancefactor involved in GBS pathogenicity and is therefore useful as anactive agent in compositions for preventing and for treating S.agalactiae infections.

I. BibA Polypeptides

“BibA polypeptides” of the invention comprise a portion of a BibAprotein which consists of (1) a coiled-coil domain of the BibA protein;(2) a leader sequence and the coiled-coil domain of the BibA protein;(3) a proline-rich domain of the BibA protein; (4) the coiled-coil andproline-rich domains of the BibA protein; or (5) the leader sequence,the coiled-coil domain, and the proline-rich domain of the BibA proteinand are free of other contiguous amino acid sequences of the BibAprotein. BibA polypeptides of the invention do not comprise the aminoacid sequence of a full-length BibA polypeptide.

BibA polypeptides include those polypeptides identified as “I,” “II,”and “III” in FIG. 24.

BibA protein from GBS serotype V isolated strain 2603 V/R has the aminoacid sequence shown in SEQ ID NO:1:

MNNNEKKVKYFLRKTAYGLASMSAAFAVCSGIVHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESIKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKK

EAASPLLA IVSLIVMLSAGLITIVLKHKKN

BibA contains an N-terminal leader or signal sequence domain which isindicated by the underlined sequence at the beginning of SEQ ID NO:1above and the C-terminal transmembrane domain which is indicated by theunderlined sequence at the end of SEQ ID NO:1 above. One or more aminoacids from the leader or signal sequence domain of BibA may be removed.An example of such a BibA fragment is set forth below as SEQ ID NO:2:

TSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKKLPSTGEAASPLLAIVSLIVMLSAGLITIVLKHKKN

BibA also contains an amino acid motif indicative of a cell wall anchor:

-   -   LPXTG (SEQ ID NO:3, shown in bold and italics in SEQ ID NO:1        above).

In one embodiment, the leader or signal sequence domain, thetransmembrane and cytoplasmic domains, and the cell wall anchor motifare removed from the BibA sequence to leave the coiled-coil andproline-rich segments as set forth below as SEQ ID NO:4:

TSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKK

The proline-rich domain of BibA is indicated below as SEQ ID NO:5.

PDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKFEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKK

The coiled-coil domain and signal peptide domain of BibA are set forthbelow as SEQ ID NO:6:

MNNNEKKVKYFLRKTAYGLASMSAAFAVCSGIVHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAK

The highly conserved coiled-coil domain of BibA is located towards theN-terminus of the protein and is underlined in the BibA SEQ ID NO:1sequence below. The underlined fragment corresponding to the coiled-coildomain of BibA is set forth below as SEQ ID NO:7:

SEQ ID NO: 1MNNNEKKVKYFLRKTAYGLASMSAAFAVCSGIVHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKKLPSTGEAASPLLAIVSLIVMLSAGLITIVLKHKKN SEQ ID NO: 7TSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGPISSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKK

The signal peptide (amino acids 1-36), coiled coil domain, andproline-rich domain of BibA are set forth below in SEQ ID NO:8:

MNNNEKKVKYFLRKTAYGLASMSAAFAVCSGIVHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKK

II. Nucleic Acid Molecules Encoding BibA Polypeptides

The invention includes nucleic acid molecules which encode BibApolypeptides. The invention also includes nucleic acid moleculescomprising nucleotide sequences having at least 50% sequence identity tosuch molecules. Depending on the particular sequence, the degree ofsequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).Identity between nucleotide sequences is preferably determined by theSmith-Waterman homology search algorithm as implemented in the MPSRCHprogram (Oxford Molecular), using an affine gap search with parametersgap open penalty=12 and gap extension penalty=1.

The invention also provides nucleic acid molecules which can hybridizeto these molecules. Hybridization reactions can be performed underconditions of different “stringency.” Conditions which increasestringency of a hybridization reaction are widely known and published inthe art. See, e.g., page 7.52 of Sambrook et al., Molecular Cloning: ALaboratory Manual, 1989. Examples of relevant conditions include (inorder of increasing stringency): incubation temperatures of 25° C., 37°C., 50° C., 55° C., and 68° C.; buffer concentrations of 10×SSC, 6×SSC,1×SSC, and 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer)and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, orde-ionized water. Hybridization techniques and their optimization arewell known in the art. See, e.g., Sambrook, 1989; Ausubel et al., eds.,Short Protocols in Molecular Biology, 4th ed., 1999; U.S. Pat. No.5,707,829; Ausubel et al., eds., Current Protocols in Molecular Biology,Supplement 30, 1987.

In some embodiments, nucleic acid molecules of the invention hybridizeto a target under low stringency conditions; in other embodiments,nucleic acid molecules of the invention hybridize under intermediatestringency conditions; in preferred embodiments, nucleic acid moleculesof the invention hybridize under high stringency conditions. An exampleof a low stringency hybridization condition is 50° C. and 10×SSC. Anexample of an intermediate stringency hybridization condition is 55° C.and 1×SSC. An example of a high stringency hybridization condition is68° C. and 0.1×SSC.

Nucleic acid molecules comprising fragments of these sequences are alsoincluded in the invention. These comprise at least n consecutivenucleotides of these sequences and, depending on the particularsequence, n is 10 or more (e.g., 12, 14, 15, 18, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100, 150, 200, or more).

Nucleic acids (and polypeptides) of the invention may include sequenceswhich:

-   -   (a) are identical (i.e., 100% identical) to the sequences        disclosed in the sequence listing;    -   (b) share sequence identity with the sequences disclosed in the        sequence listing;    -   (c) have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 single nucleotide or        amino acid alterations (deletions, insertions, substitutions),        which may be at separate locations or may be contiguous, as        compared to the sequences of (a) or (b); and,    -   (d) when aligned with a particular sequence from the sequence        listing using a pairwise alignment algorithm, a moving window of        x monomers (amino acids or nucleotides) moving from start        (N-terminus or 5′) to end (C-terminus or 3′), such that for an        alignment that extends to p monomers (where p>x) there are p-x+1        such windows, each window has at least x·y identical aligned        monomers, where: x is selected from 20, 25, 30, 35, 40, 45, 50,        60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60,        0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95,        0.96, 0.97, 0.98, 0.99; and if x·y is not an integer then it is        rounded up to the nearest integer. The preferred pairwise        alignment algorithm is the Needleman-Wunsch global alignment        algorithm [Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453],        using default parameters (e.g., with Gap opening penalty=10.0,        and with Gap extension penalty=0.5, using the EBLOSUM62 scoring        matrix). This algorithm is conveniently implemented in the        needle tool in the EMBOSS package [Rice et al. (2000) Trends        Genet. 16:276-277].

The nucleic acids and polypeptides of the invention may additionallyhave further sequences to the N-terminus/5′ and/or C-terminus/3′ ofthese sequences (a) to (d).

Nucleic acid molecules of the invention can be single- ordouble-stranded and can be used, for example, to produce BibApolypeptides in vitro (i.e., a recombinant protein) or in vivo (as a DNAvaccine). The invention also provides single-stranded nucleic acidmolecules which can hybridize to other nucleic acid molecules of theinvention, preferably under “high stringency” conditions (e.g., 65° C.in a 0.1×SSC, 0.5% SDS solution).

Nucleic acid molecules of the invention can comprise DNA or RNA,including analogues, such as those containing modified backbones (e.g.,phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc.Nucleic acid molecules of the invention can comprise portions of genomicDNA, cDNA, or mRNA. Nucleic acid molecules of the invention do notencode full-length BibA proteins.

An example of a nucleic acid molecule which encodes a full-length BibAprotein from which portions encoding BibA polypeptides can be derived isset forth below as SEQ ID NO:16:

ATGAATAATAACGAAAAAAAAGTAAAATACTTTTTAAGAAAAACAGCTTATGGTTTGGCCTCAATGTCAGCAGCGTTTGCTGTATGTAGTGGTATTGTACACGCGGATACTAGTTCAGGAATATCGGCTTCAATTCCTCATAAGAAACAAGTTAATTTAGGGGCGGTTACTCTGAAGAATTTGATTTCTAAATATCGTGGTAATGACAAAGCTATTGCTATACTTTTAAGTAGAGTAAATGATTTTAATAGAGCATCACAGGATACACTTCCACAATTAATTAATAGTACTGAAGCAGAAATTAGAAATATTTTATATCAAGGACAAATTGGTAAGCAAAATAAACCAAGTGTAACTACACATGCTAAAGTTAGTGATCAAGAACTAGGTAAGCAGTCAAGACGTTCTCAAGATATCATTAAGTCATTAGGTTTCCTTTCATCAGACCAAAAAGATATTTTAGTTAAATCTATTAGCTCTTCAAAAGATTCGCAACTTATTCTTAAATTTGTAACTCAAGCCACGCAACTGAATAATGCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGCAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGCAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCAGAGGTACTAAAGAAGATAAAAAACCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGAAGCTAAGCCAGAAGCTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAACCAGAGGCTAAGCCAGAAGCTAAACCAGAGGCTAAGTCAGAAGCTAAACCAGAGGCTAAGCTAGAAGCTAAACCAGAGGCCAAACCAGCAACCAAAAAATCGGTTAATACTAGCGGAAACTTGGCGGCTAAAAAAGCTATTGAAAACAAAAAGTATAGTAAAAAATTACCATCAACGGGTGAAGCCGCAAGTCCACTCTTAGCAATTGTATCACTAATTGTTATGTTAAGTGCAGGTCTTATTACGATAGTTTTAAAGCATAAAAAAAAT

Other embodiments of the invention provide nucleic acid molecules whichencode a proline-rich domain of a BibA polypeptide. An example of such anucleic acid molecule is set forth below as SEQ ID NO:17:

CCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGAAGCTAAGCCAGAAGCTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAACCAGAGGCTAAGCCAGAAGCTAAACCAGAGGCTAAGTCAGAAGCTAAACCAGAGGCTAAGCTAGAAGCTAAACCAGAGGCCAAACCAGCAACCAAAAAATCGGTTAATACTAGCGGAAACTTGGCGGCTAAAAAAGCTATTGAAAACAAAAAGTATAGTAAAAAA

A nucleic acid molecule encoding a highly conserved coiled-coil domainand proline-rich domain of a BibA polypeptide is set forth below as SEQID NO:18:

GGTATTGTACACGCGGATACTAGTTCAGGAATATCGGCTTCAATTCCTCATAAGAAACAAGTTAATTTAGGGGCGGTTACTCTGAAGAATTTGATTTCTAAATATCGTGGTAATGACAAAGCTATTGCTATACTTTTAAGTAGAGTAAATGATTTTAATAGAGCATCACAGGATACACTTCCACAATTAATTAATAGTACTGAAGCAGAAATTAGAAATATTTTATATCAAGGACAAATTGGTAAGCAAAATAAACCAAGTGTAACTACACATGCTAAAGTTAGTGATCAAGAACTAGGTAAGCAGTCAAGACGTTCTCAAGATATCATTAAGTCATTAGGTTTCCTTTCATCAGACCAAAAAGATATTTTAGTTAAATCTATTAGCTCTTCAAAAGATTCGCAACTTATTCTTAAATTTGTAACTCAAGCCACGCAACTGAATAATGCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGCAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGCAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCAGAGGTACTAAAGAAGATAAAAAACCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAA

A nucleic acid molecule which encodes a cell wall anchor of BibA is setforth as SEQ ID NO:19: TTACCATCAACGGGT.

III. Preparation of Nucleic Acid Molecules

Nucleic acid molecules of the invention can be prepared in many ways,for example, by chemical synthesis, from genomic or cDNA libraries(e.g., using primer-based amplification methods, such as PCR), from theorganism itself, etc.) and can take various forms (e.g. single-stranded,double-stranded, vectors, probes, etc.). They are preferably prepared insubstantially pure form (i.e. substantially free from other GBS or hostcell nucleic acids).

Nucleic acid molecules can be synthesized, in whole or in part, usingchemical methods well known in the art. See Caruthers et al., Nucl.Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp.Ser. 225-232, 1980; Hunkapiller et al. (1984), Nature 310: 105-111;Grantham et al. (1981), Nucleic Acids Res. 9: r43-r74.

cDNA molecules can be made with standard molecular biology techniques,using mRNA as a template. cDNA molecules can thereafter be replicatedusing molecular biology techniques well known in the art. Anamplification technique, such as PCR, can be used to obtain additionalcopies of polynucleotides of the invention, using either genomic DNA orcDNA as a template.

If desired, nucleotide sequences can be engineered using methodsgenerally known in the art to alter coding sequences for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the polypeptide or mRNAproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides can be used to engineerthe nucleotide sequences. For example, site-directed mutagenesis can beused to insert new restriction sites, alter glycosylation patterns,change codon preference, produce splice variants, introduce mutations,and so forth.

Sequence modifications, such as the addition of a purification tagsequence or codon optimization, can be used to facilitate expression.For example, the N-terminal leader sequence may be replaced with asequence encoding for a tag protein such as polyhistidine (“HIS”) orglutathione S-transferase (“GST”). Such tag proteins may be used tofacilitate purification, detection, and stability of the expressedprotein. Codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producean RNA transcript having desirable properties, such as a half-life whichis longer than that of a transcript generated from the naturallyoccurring sequence. These methods are well known in the art and arefurther described in WO05/032582.

IV. Production of BibA Polypeptides

BibA polypeptides can be produced recombinantly, for example, byculturing a host cell transformed with nucleic acid molecules of theinvention under conditions which permit polypeptide expression. BibApolypeptides can be synthesized by chemical means, or can be preparedfrom full-length BibA protein isolated from S. agalactiae.

A. Recombinant Production of Polypeptides

1. Nucleic Acid Molecules

Any nucleic acid molecule which encodes a particular BibA polypeptidecan be used to produce that polypeptide recombinantly. Recombinantproduction of BibA polypeptides can be facilitated by adding anucleotide sequence encoding a tag protein in frame to the nucleotidesequence encoding the BibA polypeptide such that the polypeptide isexpressed as a fusion protein comprising the tag protein and the GBSpolypeptide. Such tag proteins can facilitate purification, detection,and stability of the expressed protein. Tag proteins suitable for use inthe invention include a polyarginine tag (Arg-tag), polyhistidine tag(His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-bindingpeptide, cellulose-binding domain, SBP-tag, chitin-binding domain,glutathione S-transferase-tag (GST), maltose-binding protein,transcription termination anti-termination factor (NusA), E. colithioredoxin (TrxA), and protein disulfide isomerase I (DsbA). Preferredtag proteins include His-tag and GST. See Terpe et al., Appl MicrobiolBiotechnol (2003) 60:523-33.

After purification, a tag protein may optionally be removed from theexpressed fusion protein, i.e., by specifically tailored enzymatictreatments known in the art. Commonly used proteases includeenterokinase, tobacco etch virus (TEV), thrombin, and factor X_(a).

2. Expression Constructs

A nucleic acid molecule which encodes a polypeptide can be inserted intoan expression construct which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodswhich are well known to those skilled in the art can be used toconstruct expression constructs containing coding sequences andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination.

3. Host Cells

The heterologous host can be prokaryotic or eukaryotic. E. coli is apreferred host cell, but other suitable hosts include Bacillus subtilis,Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisserialactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis),yeasts, etc.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedpolypeptide in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of thepolypeptide also can be used to facilitate correct insertion, foldingand/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities are available from the American Type Culture Collection(ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can bechosen to ensure the correct modification and processing of a foreignprotein. See WO 01/98340.

Expression constructs can be introduced into host cells usingwell-established techniques which include, but are not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun” methods, and DEAE-or calcium phosphate-mediated transfection.

Host cells transformed with expression constructs can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The protein produced by a transformed cell can be secretedor contained intracellularly depending on the nucleotide sequence and/orthe expression construct used. Those of skill in the art understand thatexpression constructs can be designed to contain signal sequences whichdirect secretion of soluble polypeptides through a prokaryotic oreukaryotic cell membrane.

B. Purification

BibA polypeptides of the invention can be isolated from the appropriateStreptococcus agalactiae bacterium or from an engineered host cell. Apurified BibA polypeptide is separated from other components in thecell, such as proteins, carbohydrates, or lipids, using methodswell-known in the art. Such methods include, but are not limited to,size exclusion chromatography, ammonium sulfate fractionation, ionexchange chromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified BibA polypeptide is at least80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purityof the preparations can be assessed by any means known in the art, suchas SDS-polyacrylamide gel electrophoresis. Where appropriate,polypeptides can be solubilized, for example, with urea.

C. Chemical Synthesis

BibA polypeptides of the invention can be synthesized, for example,using solid-phase techniques. See, e.g., Merrifield, J. Am. Chem. Soc.85, 2149-54, 1963; Roberge et al., Science 269, 202-04, 1995. Synthesiscan be performed using manual techniques or by automation. Automatedsynthesis can be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Optionally, fragments of apolypeptide can be separately synthesized and combined using chemicalmethods to produce the final molecule.

V. Antibodies

Antibodies can be generated to bind specifically to a BibA polypeptideor other antigen and can be used therapeutically and diagnostically. Theterm “antibody” includes intact immunoglobulin molecules, as well asfragments thereof which are capable of binding an antigen. These includehybrid (chimeric) antibody molecules (e.g., Winter et al., Nature 349,293-99, 1991; U.S. Pat. No. 4,816,567); F(ab′)₂ and F(ab) fragments andF_(v) molecules; non-covalent heterodimers (e.g., Inbar et al., Proc.Natl. Acad. Sci. U.S.A. 69, 2659-62, 1972; Ehrlich et al., Biochem 19,4091-96, 1980); single-chain F_(v) molecules (sFv) (e.g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A. 85, 5897-83, 1988); dimeric and trimericantibody fragment constructs; minibodies (e.g., Pack et al., Biochem 31,1579-84, 1992; Cumber et al., J. Immunology 149B, 120-26, 1992);humanized antibody molecules (e.g., Riechmann et al., Nature 332,323-27, 1988; Verhoeyan et al., Science 239, 1534-36, 1988; and U.K.Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and anyfunctional fragments obtained from such molecules, as well as antibodiesobtained through non-conventional processes such as phage display.Preferably, the antibodies are monoclonal antibodies. Methods ofobtaining monoclonal antibodies are well known in the art.

Preferred antibodies of the invention specifically bind to an epitope inthe N-terminal domain, coiled-coil domain, or proline-rich domain ofBibA. Typically, at least 6, 7, 8, 10, or 12 contiguous amino acids arerequired to form an epitope. However, epitopes which involvenon-contiguous amino acids may require more, e.g., at least 15, 25, or50 amino acids. Various immunoassays (e.g., Western blots, ELISAs,radioimmunoassays, immunohistochemical assays, immunoprecipitations, orother immunochemical assays known in the art) can be used to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays are well known in theart. Such immunoassays typically involve the measurement of complexformation between an immunogen and an antibody which specifically bindsto the immunogen. A preparation of antibodies which specifically bind toa particular antigen typically provides a detection signal at least 5-,10-, or 20-fold higher than a detection signal provided with otherproteins when used in an immunochemical assay. Preferably, theantibodies do not detect other proteins in immunochemical assays and canimmunoprecipitate the particular antigen from solution.

Polypeptides can be used to immunize a mammal, such as a mouse, rat,rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.If desired, an antigen can be conjugated to a carrier protein, such asbovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.Depending on the host species, various adjuvants can be used to increasethe immunological response. Such adjuvants include, but are not limitedto, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol). Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to an antigen can beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These techniquesinclude, but are not limited to, the hybridoma technique, the humanB-cell hybridoma technique, and the EBV-hybridoma technique (Kohler etal., Nature 256, 495-497, 1985; Kozbor et al., J. Immunol. Methods 81,31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983;Cole et al., Mol. Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984;Takeda et al., Nature 314, 452-454, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.

Alternatively, humanized antibodies can be produced using recombinantmethods, as described below. Antibodies which specifically bind to aparticular antigen can contain antigen binding sites which are eitherpartially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to a particular antigen.Antibodies with related specificity, but of distinct idiotypiccomposition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88,11120-23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, 1994, J. Biol.Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., 1995,Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth.165, 81-91).

Antibodies which specifically bind to a particular antigen also can beproduced by inducing in vivo production in the lymphocyte population orby screening immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in the literature (Orlandi et al., Proc.Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349,293-299, 1991).

Chimeric antibodies can be constructed as disclosed in WO 93/03151.Binding proteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared.

Antibodies can be purified by methods well known in the art. Forexample, antibodies can be affinity purified by passage over a column towhich the relevant antigen is bound. The bound antibodies can then beeluted from the column using a buffer with a high salt concentration.

VI. Compositions Comprising One or More Active Agents

The invention provides compositions for preventing and for treating S.agalactiae infection. Compositions of the invention comprise at leastone active agent. The active agent can be a BibA polypeptide, a nucleicacid molecule encoding a BibA polypeptide, or antibodies whichspecifically bind to a BibA polypeptide. Suitable BibA polypeptidesinclude, for example, those identified in groups “I,” “II,” and “III” inFIG. 24. Compositions of the invention can include one or more BibApolypeptides of two or more of these groups.

Compositions of the invention can include one or more additional activeagents. Such agents include, but are not limited to one or more (a) GBSantigens, (b) non-GBS antigens, (c) nucleic acid molecules encoding (a)or (b), and antibodies which specifically bind to (a) or (b).

A. GBS Antigens

GBS antigens which can be included in compositions of the inventioninclude antigenic portions of the GBS proteins disclosed in WO 02/34771(e.g., GBS1-GBS689), which is incorporated herein by reference in itsentirety. Preferred antigens include GBS 80, GBS 104, GBS 322, GBS 67,GBS 276, and GBS 59.

B. Non-GBS Antigens

Compositions of the invention may be administered in conjunction withone or more antigens for use in therapeutic, prophylactic, or diagnosticmethods of the present invention. Compositions of the inventionoptionally can comprise one or more additional polypeptide antigenswhich are not derived from S. agalactiae proteins. Preferred antigensinclude those listed below. Additionally, the compositions of thepresent invention may be used to treat or prevent infections caused byany of the below-listed pathogens. In addition to combination with theantigens described below, the compositions of the invention may also becombined with an adjuvant as described herein.

Antigens for use with the invention include, but are not limited to, oneor more of the following antigens set forth below, or antigens derivedfrom one or more of the pathogens set forth below:

1. Bacterial Antigens

Bacterial antigens suitable for use in the invention include proteins,polysaccharides, lipopolysaccharides, and outer membrane vesicles whichmay be isolated, purified or derived from a bacteria. In addition,bacterial antigens may include bacterial lysates and inactivatedbacteria formulations. Bacteria antigens may be produced by recombinantexpression. Bacterial antigens preferably include epitopes which areexposed on the surface of the bacteria during at least one stage of itslife cycle. Bacterial antigens are preferably conserved across multipleserotypes. Bacterial antigens include antigens derived from one or moreof the bacteria set forth below as well as the specific antigensexamples identified below.

Neisseria meningitides: Meningitides antigens may include proteins (suchas those identified in References 1-7), saccharides (including apolysaccharide, oligosaccharide or lipopolysaccharide), orouter-membrane vesicles (References 8, 9, 10, 11) purified or derivedfrom N. meningitides serogroup such as A, C, W135, Y, and/or B.Meningitides protein antigens may be selected from adhesions,autotransporters, toxins, Fe acquisition proteins, and membraneassociated proteins (preferably integral outer membrane protein).

Streptococcus pneumoniae: Streptococcus pneumoniae antigens may includea saccharide (including a polysaccharide or an oligosaccharide) and/orprotein from Streptococcus pneumoniae. Saccharide antigens may beselected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Protein antigens maybe selected from a protein identified in WO 98/18931, WO 98/18930, U.S.Pat. No. 6,699,703, U.S. Pat. No. 6,800,744, WO 97/43303, and WO97/37026. Streptococcus pneumoniae proteins may be selected from thePoly Histidine Triad family (PhtX), the Choline Binding Protein family(CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytXtruncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101,Sp130, Sp125 or Sp133.

Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcusantigens may include a protein identified in WO 02/34771 or WO2005/032582 (including GBS 40), fusions of fragments of GBS M proteins(including those described in WO 02/094851, and Dale, Vaccine (1999)17:193-200, and Dale, Vaccine 14(10): 944-948), fibronectin bindingprotein (Sfb1), Streptococcal heme-associated protein (Shp), andStreptolysin S (SagA).

Moraxella catarrhalis: Moraxella antigens include antigens identified inWO 02/18595 and WO 99/58562, outer membrane protein antigens (HMW-OMP),C-antigen, and/or LPS.

Bordetella pertussis: Pertussis antigens include pertussis holotoxin(PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionallyalso combination with pertactin and/or agglutinogens 2 and 3 antigen.

Staphylococcus aureus: Staphylococcus aureus antigens include S. aureustype 5 and 8 capsular polysaccharides optionally conjugated to nontoxicrecombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, orantigens derived from surface proteins, invasins (leukocidin, kinases,hyaluronidase), surface factors that inhibit phagocytic engulfment(capsule, Protein A), carotenoids, catalase production, Protein A,coagulase, clotting factor, and/or membrane-damaging toxins (optionallydetoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin,leukocidin).

Staphylococcus epidermis: S. epidermidis antigens includeslime-associated antigen (SAA).

Clostridium tetani (Tetanus): Tetanus antigens include tetanus toxoid(TT), preferably used as a carrier protein in conjunction/conjugatedwith the compositions of the present invention.

Cornynebacterium diphtheriae (Diphtheria): Diphtheria antigens includediphtheria toxin, preferably detoxified, such as CRM197. Additionallyantigens capable of modulating, inhibiting or associated with ADPribosylation are contemplated forcombination/co-administration/conjugation with the compositions of thepresent invention. The diphtheria toxoids may be used as carrierproteins.

Haemophilus influenzae B (Hib): Hib antigens include a Hib saccharideantigen.

Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzzprotein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5serotype), and/or Outer Membrane Proteins, including Outer MembraneProteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515).

Legionella pneumophila. Bacterial antigens may be derived fromLegionella pneumophila.

Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcusantigens include a protein or saccharide antigen identified in WO02/34771, WO 03/093306, WO 04/041157, or WO 2005/002619 (includingproteins GBS 80, GBS 104, GBS 276 and GBS 322, and including saccharideantigens derived from serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VIIand VIII).

Neiserria gonorrhoeae: Gonorrhoeae antigens include Por (or porin)protein, such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), atransferring binding protein, such as TbpA and TbpB (See Price et al.,Infection and Immunity (2004) 71(1):277-283), a opacity protein (such asOpa), a reduction-modifiable protein (Rmp), and outer membrane vesicle(OMV) preparations (see Plante et al., J Infectious Disease (2000)182:848-855), also see e.g. WO99/24578, WO99/36544, WO99/57280,WO02/079243).

Chlamydia trachomatis: Chlamydia trachomatis antigens include antigensderived from serotypes A, B, Ba and C (agents of trachoma, a cause ofblindness), serotypes L1, L2 & L3 (associated with Lymphogranulomavenereum), and serotypes, D-K. Chlamydia trachomas antigens may alsoinclude an antigen identified in WO 00/37494, WO 03/049762, WO03/068811, or WO 05/002619, including PepA (CT045), LcrE (CT089), ArtJ(CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA(CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG(CT761).

Treponema pallidum (Syphilis): Syphilis antigens include TmpA antigen.

Haemophilus ducreyi (causing chancroid): Ducreyi antigens include outermembrane protein (DsrA).

Enterococcus faecalis or Enterococcus faecium: Antigens include atrisaccharide repeat or other Enterococcus derived antigens provided inU.S. Pat. No. 6,756,361.

Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX,HopY and/or urease antigen.

Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutininof S. saprophyticus antigen.

Yersinia enterocolitica antigens include LPS (Infect Immun. 2002 August;70(8): 4414).

E. coli: E. coli antigens may be derived from enterotoxigenic E. coli(ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli(DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E.coli (EHEC).

Bacillus anthracis (anthrax): B. anthracis antigens are optionallydetoxified and may be selected from A-components (lethal factor (LF) andedema factor (EF)), both of which can share a common B-component knownas protective antigen (PA).

Yersinia pestis (plague): Plague antigens include F1 capsular antigen(Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun. 1999October; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997November; 65(11): 4476-4482).

Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins,LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6optionally formulated in cationic lipid vesicles (Infect Immun. 2004October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitratedehydrogenase associated antigens (Proc Natl Acad Sci USA. 2004 Aug. 24;101(34): 12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7):3829).

Rickettsia: Antigens include outer membrane proteins, including theouter membrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004Nov. 1; 1702(2):145), LPS, and surface protein antigen (SPA) (J.Autoimmun. 1989 June; 2 Suppl:81).

Listeria monocytogenes. Bacterial antigens may be derived from Listeriamonocytogenes.

Chlamydia pneumoniae: Antigens include those identified in WO 02/02606.

Vibrio cholerae: Antigens include proteinase antigens, LPS, particularlylipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specificpolysaccharides, V. cholera 0139, antigens of IEM108 vaccine (InfectImmun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin(Zot).

Salmonella typhi (typhoid fever): Antigens include capsularpolysaccharides preferably conjugates (Vi, i.e. vax-TyVi).

Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (suchas OspA, OspB, Osp C and Osp D), other surface proteins such asOspE-related proteins (Erps), decorin-binding proteins (such as DbpA),and antigenically variable VI proteins, such as antigens associated withP39 and P13 (an integral membrane protein, Infect Immun. 2001 May;69(5): 3323-3334), VlsE Antigenic Variation Protein (J Clin Microbiol.1999 December; 37(12): 3997).

Porphyromonas gingivalis: Antigens include P. gingivalis outer membraneprotein (OMP).

Klebsiella: Antigens include an OMP, including OMP A, or apolysaccharide optionally conjugated to tetanus toxoid.

Further bacterial antigens of the invention may be capsular antigens,polysaccharide antigens or protein antigens of any of the above. Furtherbacterial antigens may also include an outer membrane vesicle (OMV)preparation. Additionally, antigens include live, attenuated, and/orpurified versions of any of the aforementioned bacteria. The antigens ofthe present invention may be derived from gram-negative or gram-positivebacteria. The antigens of the present invention may be derived fromaerobic or anaerobic bacteria.

Additionally, any of the above bacterial-derived saccharides(polysaccharides, LPS, LOS or oligosaccharides) can be conjugated toanother agent or antigen, such as a carrier protein (for exampleCRM197). Such conjugation may be direct conjugation effected byreductive amination of carbonyl moieties on the saccharide to aminogroups on the protein, as provided in U.S. Pat. No. 5,360,897 and Can JBiochem Cell Biol. 1984 May; 62(5):270-5. Alternatively, the saccharidescan be conjugated through a linker, such as, with succinamide or otherlinkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry ofProtein Conjugation and Cross-Linking, 1993.

2. Viral Antigens

Viral antigens suitable for use in the invention include inactivated (orkilled) virus, attenuated virus, split virus formulations, purifiedsubunit formulations, viral proteins which may be isolated, purified orderived from a virus, and Virus Like Particles (VLPs). Viral antigensmay be derived from viruses propagated on cell culture or othersubstrate. Alternatively, viral antigens may be expressed recombinantly.Viral antigens preferably include epitopes which are exposed on thesurface of the virus during at least one stage of its life cycle. Viralantigens are preferably conserved across multiple serotypes or isolates.Viral antigens include antigens derived from one or more of the virusesset forth below as well as the specific antigens examples identifiedbelow.

Orthomyxovirus: Viral antigens may be derived from an Orthomyxovirus,such as Influenza A, B and C. Orthomyxovirus antigens may be selectedfrom one or more of the viral proteins, including hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membraneprotein (M2), one or more of the transcriptase components (PB1, PB2 andPA). Preferred antigens include HA and NA.

Influenza antigens may be derived from interpandemic (annual) flustrains. Alternatively influenza antigens may be derived from strainswith the potential to cause pandemic a pandemic outbreak (i.e.,influenza strains with new haemagglutinin compared to the haemagglutininin currently circulating strains, or influenza strains which arepathogenic in avian subjects and have the potential to be transmittedhorizontally in the human population, or influenza strains which arepathogenic to humans).

Paramyxoviridae viruses: Viral antigens may be derived fromParamyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses(PIV) and Morbilliviruses (Measles).

Pneumovirus: Viral antigens may be derived from a Pneumovirus, such asRespiratory syncytial virus (RSV), Bovine respiratory syncytial virus,Pneumonia virus of mice, and Turkey rhinotracheitis virus. Preferably,the Pneumovirus is RSV. Pneumovirus antigens may be selected from one ormore of the following proteins, including surface proteins Fusion (F),Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins Mand M2, nucleocapsid proteins N, P and L and nonstructural proteins NS1and NS2. Preferred Pneumovirus antigens include F, G and M. See e.g., JGen Virol. 2004 November; 85(Pt 11):3229). Pneumovirus antigens may alsobe formulated in or derived from chimeric viruses. For example, chimericRSV/PIV viruses may comprise components of both RSV and PIV.

Paramyxovirus: Viral antigens may be derived from a Paramyxovirus, suchas Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simianvirus 5, Bovine parainfluenza virus and Newcastle disease virus.Preferably, the Paramyxovirus is PIV or Mumps. Paramyxovirus antigensmay be selected from one or more of the following proteins:Hemagglutinin-Neuraminidase (HN), Fusion proteins F1 and F2,Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrixprotein (M). Preferred Paramyxovirus proteins include HN, F1 and F2.Paramyxovirus antigens may also be formulated in or derived fromchimeric viruses. For example, chimeric RSV/PIV viruses may comprisecomponents of both RSV and PIV. Commercially available mumps vaccinesinclude live attenuated mumps virus, in either a monovalent form or incombination with measles and rubella vaccines (MMR).

Morbillivirus: Viral antigens may be derived from a Morbillivirus, suchas Measles. Morbillivirus antigens may be selected from one or more ofthe following proteins: hemagglutinin (H), Glycoprotein (G), Fusionfactor (F), Large protein (L), Nucleoprotein (NP), Polymerasephosphoprotein (P), and Matrix (M). Commercially available measlesvaccines include live attenuated measles virus, typically in combinationwith mumps and rubella (MMR).

Picornavirus: Viral antigens may be derived from Picornaviruses, such asEnteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses andAphthoviruses. Antigens derived from Enteroviruses, such as Poliovirusare preferred.

Enterovirus: Viral antigens may be derived from an Enterovirus, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71. Preferably, the Enterovirusis poliovirus. Enterovirus antigens are preferably selected from one ormore of the following Capsid proteins VP1, VP2, VP3 and VP4.Commercially available polio vaccines include Inactivated Polio Vaccine(IPV) and Oral poliovirus vaccine (OPV).

Heparnavirus: Viral antigens may be derived from an Heparnavirus, suchas Hepatitis A virus (HAV). Commercially available HAV vaccines includeinactivated HAV vaccine.

Togavirus: Viral antigens may be derived from a Togavirus, such as aRubivirus, an Alphavirus, or an Arterivirus. Antigens derived fromRubivirus, such as Rubella virus, are preferred. Togavirus antigens maybe selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirusantigens are preferably selected from E1, E2 or E3. Commerciallyavailable Rubella vaccines include a live cold-adapted virus, typicallyin combination with mumps and measles vaccines (MMR).

Flavivirus: Viral antigens may be derived from a Flavivirus, such asTick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), YellowFever, Japanese encephalitis, West Nile encephalitis, St. Louisencephalitis, Russian spring-summer encephalitis, Powassan encephalitis.Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a,NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferablyselected from PrM, M and E. Commercially available TBE vaccine includeinactivated virus vaccines.

Pestivirus: Viral antigens may be derived from a Pestivirus, such asBovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Borderdisease (BDV).

Hepadnavirus: Viral antigens may be derived from a Hepadnavirus, such asHepatitis B virus. Hepadnavirus antigens may be selected from surfaceantigens (L, M and S), core antigens (HBc, HBe). Commercially availableHBV vaccines include subunit vaccines comprising the surface antigen Sprotein.

Hepatitis C virus: Viral antigens may be derived from a Hepatitis Cvirus (HCV). HCV antigens may be selected from one or more of E1, E2,E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptidesfrom the nonstructural regions (Houghton et al., Hepatology (1991)14:381).

Rhabdovirus: Viral antigens may be derived from a Rhabdovirus, such as aLyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigensmay be selected from glycoprotein (G), nucleoprotein (N), large protein(L), nonstructural proteins (NS). Commercially available Rabies virusvaccine comprise killed virus grown on human diploid cells or fetalrhesus lung cells.

Caliciviridae; Viral antigens may be derived from Calciviridae, such asNorwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and SnowMountain Virus.

Coronavirus: Viral antigens may be derived from a Coronavirus, SARS,Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mousehepatitis virus (MHV), and Porcine transmissible Gastroenteritis virus(TGEV). Coronavirus antigens may be selected from spike (S), envelope(E), matrix (M), nucleocapsid (N), and Hemagglutinin-esteraseglycoprotein (HE). Preferably, the Coronavirus antigen is derived from aSARS virus. SARS viral antigens are described in WO 04/92360;

Retrovirus: Viral antigens may be derived from a Retrovirus, such as anOncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may bederived from HTLV-1, HTLV-2 or HTLV-5. Lentivirus antigens may bederived from HIV-1 or HIV-2. Retrovirus antigens may be selected fromgag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigensmay be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol,tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete).HIV antigens may be derived from one or more of the following strains:HIVIIIb, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV-1CM235, HIV-1US4.

Reovirus: Viral antigens may be derived from a Reovirus, such as anOrthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Reovirusantigens may be selected from structural proteins λ1, λ2, λ3, μ1, μ2,σ1, σ2, or σ3, or nonstructural proteins σNS, μNS, or σ1s. PreferredReovirus antigens may be derived from a Rotavirus. Rotavirus antigensmay be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 andVP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred Rotavirusantigens include VP4 (or the cleaved product VP5 and VP8), and VP7.

Parvovirus: Viral antigens may be derived from a Parvovirus, such asParvovirus B19. Parvovirus antigens may be selected from VP-1, VP-2,VP-3, NS-1 and NS-2. Preferably, the Parvovirus antigen is capsidprotein VP-2.

Delta hepatitis virus (HDV): Viral antigens may be derived HDV,particularly δ-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).

Hepatitis E virus (HEV): Viral antigens may be derived from HEV.

Hepatitis G virus (HGV): Viral antigens may be derived from HGV.

Human Herpesvirus: Viral antigens may be derived from a HumanHerpesvirus, such as Herpes Simplex Viruses (HSV), Varicella-zostervirus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), HumanHerpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus8 (HHV8). Human Herpesvirus antigens may be selected from immediateearly proteins (a), early proteins (β), and late proteins (γ). HSVantigens may be derived from HSV-1 or HSV-2 strains. HSV antigens may beselected from glycoproteins gB, gC, gD and gH, fusion protein (gB), orimmune escape proteins (gC, gE, or gI). VZV antigens may be selectedfrom core, nucleocapsid, tegument, or envelope proteins. A liveattenuated VZV vaccine is commercially available. EBV antigens may beselected from early antigen (EA) proteins, viral capsid antigen (VCA),and glycoproteins of the membrane antigen (MA). CMV antigens may beselected from capsid proteins, envelope glycoproteins (such as gB andgH), and tegument proteins

Papovaviruses: Antigens may be derived from Papovaviruses, such asPapillomaviruses and Polyomaviruses. Papillomaviruses include HPVserotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47,51, 57, 58, 63 and 65. Preferably, HPV antigens are derived fromserotypes 6, 11, 16 or 18. HPV antigens may be selected from capsidproteins (L1) and (L2), or E1-E7, or fusions thereof. HPV antigens arepreferably formulated into virus-like particles (VLPs). Polyomyavirusviruses include BK virus and JK virus. Polyomavirus antigens may beselected from VP1, VP2 or VP3.

Further provided are antigens, compositions, methods, and microbesincluded in Vaccines, 4th Edition (Plotkin and Orenstein ed. 2004);Medical Microbiology 4th Edition (Murray et al. ed. 2002); Virology, 3rdEdition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D. M. Knipe, eds. 1991), which are contemplated inconjunction with the compositions of the present invention.

3. Fungal Antigens

Fungal antigens for use in the invention may be derived from one or moreof the fungi set forth below.

Fungal antigens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme.

Fungal pathogens may be derived from Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystiscarinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomycescerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporiumapiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasmagondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp.,Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp.,Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamellaspp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporiumspp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp,Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

Processes for producing a fungal antigens are well known in the art (seeU.S. Pat. No. 6,333,164). In a preferred method a solubilized fractionextracted and separated from an insoluble fraction obtainable fromfungal cells of which cell wall has been substantially removed or atleast partially removed, characterized in that the process comprises thesteps of: obtaining living fungal cells; obtaining fungal cells of whichcell wall has been substantially removed or at least partially removed;bursting the fungal cells of which cell wall has been substantiallyremoved or at least partially removed; obtaining an insoluble fraction;and extracting and separating a solubilized fraction from the insolublefraction.

4. STD Antigens

The compositions of the invention may include one or more antigensderived from a sexually transmitted disease (STD). Such antigens mayprovide for prophylactis or therapy for STD's such as chlamydia, genitalherpes, hepatitis (such as HCV), genital warts, gonorrhoea, syphilisand/or chancroid (See, WO00/15255). Antigens may be derived from one ormore viral or bacterial STD's. Viral STD antigens for use in theinvention may be derived from, for example, HIV, herpes simplex virus(HSV-1 and HSV-2), human papillomavirus (HPV), and hepatitis (HCV).Bacterial STD antigens for use in the invention may be derived from, forexample, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponemapallidum, Haemophilus ducreyi, E. coli, and Streptococcus agalactiae.Examples of specific antigens derived from these pathogens are describedabove.

5. Respiratory Antigens

The compositions of the invention may include one or more antigensderived from a pathogen which causes respiratory disease. For example,respiratory antigens may be derived from a respiratory virus such asOrthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus (PIV),Morbillivirus (measles), Togavirus (Rubella), VZV, and Coronavirus(SARS). Respiratory antigens may be derived from a bacteria which causesrespiratory disease, such as Streptococcus pneumoniae, Pseudomonasaeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasmapneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxellacatarrhalis. Examples of specific antigens derived from these pathogensare described above.

6. Pediatric Vaccine Antigens

The compositions of the invention may include one or more antigenssuitable for use in pediatric subjects. Pediatric subjects are typicallyless than about 3 years old, or less than about 2 years old, or lessthan about 1 years old. Pediatric antigens may be administered multipletimes over the course of 6 months, 1, 2 or 3 years. Pediatric antigensmay be derived from a virus which may target pediatric populationsand/or a virus from which pediatric populations are susceptible toinfection. Pediatric viral antigens include antigens derived from one ormore of Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), and Varicella-zoster virus(VZV), Epstein Barr virus (EBV). Pediatric bacterial antigens includeantigens derived from one or more of Streptococcus pneumoniae, Neisseriameningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridiumtetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilusinfluenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae(Group B Streptococcus), and E. coli. Examples of specific antigensderived from these pathogens are described above.

7. Antigens Suitable for Use in Elderly or Immunocompromised Individuals

The compositions of the invention may include one or more antigenssuitable for use in elderly or immunocompromised individuals. Suchindividuals may need to be vaccinated more frequently, with higher dosesor with adjuvanted formulations to improve their immune response to thetargeted antigens. Antigens which may be targeted for use in Elderly orImmunocompromised individuals include antigens derived from one or moreof the following pathogens: Neisseria meningitides, Streptococcuspneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcusepidermis, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae(Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa,Legionella pneumophila, Streptococcus agalactiae (Group BStreptococcus), Enterococcus faecalis, Helicobacter pylori, Clamydiapneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), Varicella-zoster virus(VZV), Epstein Barr virus (EBV), Cytomegalovirus (CMV). Examples ofspecific antigens derived from these pathogens are described above.

8. Antigens Suitable for Use in Adolescent Vaccines

The compositions of the invention may include one or more antigenssuitable for use in adolescent subjects. Adolescents may be in need of aboost of a previously administered pediatric antigen. Pediatric antigenswhich may be suitable for use in adolescents are described above. Inaddition, adolescents may be targeted to receive antigens derived froman STD pathogen in order to ensure protective or therapeutic immunitybefore the beginning of sexual activity. STD antigens which may besuitable for use in adolescents are described above.

9. Antigen Formulations

In other aspects of the invention, methods of producing microparticleshaving adsorbed antigens are provided. The methods comprise: (a)providing an emulsion by dispersing a mixture comprising (i) water, (ii)a detergent, (iii) an organic solvent, and (iv) a biodegradable polymerselected from the group consisting of a poly(α-hydroxy acid), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate. The polymer is typically presentin the mixture at a concentration of about 1% to about 30% relative tothe organic solvent, while the detergent is typically present in themixture at a weight-to-weight detergent-to-polymer ratio of from about0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1:1,about 0.001:1 to about 0.1:1, or about 0.005:1 to about 0.1:1); (b)removing the organic solvent from the emulsion; and (c) adsorbing anantigen on the surface of the microparticles. In certain embodiments,the biodegradable polymer is present at a concentration of about 3% toabout 10% relative to the organic solvent.

Microparticles for use herein will be formed from materials that aresterilizable, non-toxic and biodegradable. Such materials include,without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid,polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are derived from a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered macromolecule. These parameters are discussed morefully below.

Further antigens may also include an outer membrane vesicle (OMV)preparation.

Additional formulation methods and antigens (especially tumor antigens)are provided in U.S. patent Ser. No. 09/581,772.

10. Antigen References

The following references include antigens useful in conjunction with thecompositions of the present invention:

-   1 WO99/24578-   2 WO99/36544.-   3 WO99/57280.-   4 WO00/22430.-   5 Tettelin et al. (2000) Science 287:1809-1815.-   6 WO96/29412.-   7 Pizza et al. (2000) Science 287:1816-1820.-   8 PCT WO 01/52885.-   9 Bjune et al. (1991) Lancet 338(8775).-   10 Fuskasawa et al. (1999) Vaccine 17:2951-2958.-   11 Rosenqist et al. (1998) Dev. Biol. Strand 92:323-333.-   12 Constantino et al. (1992) Vaccine 10:691-698.-   13 Constantino et al. (1999) Vaccine 17:1251-1263.-   14 Watson (2000) Pediatr Infect Dis J 19:331-332.-   15 Rubin (20000) Pediatr Clin North Am 47:269-285,v.-   16 Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.-   17 filed on 3 Jul. 2001 claiming priority from GB-0016363.4; WO    02/02606; PCT IB/01/00166.-   18 Kalman et al. (1999) Nature Genetics 21:385-389.-   19 Read et al. (2000) Nucleic Acids Res 28:1397-406.-   20 Shirai et al. (2000) J. Infect. Dis 181(Suppl 3):S524-S527.-   21 WO99/27105.-   22 WO00/27994.-   23 WO00/37494.-   24 WO99/28475.-   25 Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   26 Iwarson (1995) APMIS 103:321-326.-   27 Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   28 Hsu et al. (1999) Clin Liver Dis 3:901-915.-   29 GBStofsson et al. (1996) N. Engl. J. Med. 334:349-355.-   30 Rappuoli et al. (1991) TIBTECH 9:232-238.-   31 Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0,-   32 Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.-   33 WO93/018150.-   34 WO99/53310.-   35 WO98/04702.-   36 Ross et al. (2001) Vaccine 19:135-142.-   37 Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   38 Zimmerman & Spann (1999) Am Fan Physician 59:113-118, 125-126.-   39 Dreensen (1997) Vaccine 15 Suppl″ S2-6.-   40 MMWR Morb Mortal Wkly rep 1998 January 16:47(1):12, 9.-   41 McMichael (2000) Vaccine 19 Suppl 1:S101-107.-   42 Schuchat (1999) Lancer 353(9146):51-6.-   43 GB patent applications 0026333.5, 0028727.6 & 0105640.7.-   44 Dale (1999) Infect Disclin North Am 13:227-43, viii.-   45 Ferretti et al. (2001) PNAS USA 98: 4658-4663.-   46 Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages    1218-1219.-   47 Ramsay et al. (2001) Lancet 357(9251):195-196.-   48 Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   49 Buttery & Moxon (2000) J R Coil Physicians Long 34:163-168.-   50 Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.-   51 Goldblatt (1998) J. Med. Microbiol. 47:663-567.-   52 European patent 0 477 508.-   53 U.S. Pat. No. 5,306,492.-   54 WO98/42721.-   55 Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326,    particularly vol. 10:48-114.-   56 Hermanson (1996) Bioconjugate Techniques ISBN: 012323368 &    012342335X.-   57 EP 0372501.-   58 EP 0378881.-   59 EP 0427347.-   60 WO93/17712.-   61 WO98/58668.-   62 EP 0471177.-   63 WO00/56360.-   64 WO00/67161.

The contents of all of the above cited patents, patent applications andjournal articles are incorporated by reference as if set forth fullyherein.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity. SeeRamsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond34:163-168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133,vii; Goldblatt (1998) J. Med. Microbiol. 47:563-567; European patent 0477 508; U.S. Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (eds.Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114; Hermanson(1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X. Preferredcarrier proteins are bacterial toxins or toxoids, such as diphtheria ortetanus toxoids. The CRM197 diphtheria toxoid is particularly preferred.

Other carrier polypeptides include the N. meningitidis outer membraneprotein (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A0427347), heat shock proteins (WO 93/17712 and WO 94/03208), pertussisproteins (WO 98/58668 and EP A 0471177), protein D from H. influenzae(WO 00/56360), cytokines (WO 91/01146), lymphokines, hormones, growthfactors, toxin A or B from C. difficile (WO 00/61761), iron-uptakeproteins (WO 01/72337), etc. Where a mixture comprises capsularsaccharide from both serigraphs A and C, it may be preferred that theratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g.,2:1, 3:1, 4:1, 5:1, 10:1 or higher). Different saccharides can beconjugated to the same or different type of carrier protein. Anysuitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary e.g.,detoxification of pertussis toxin by chemical and/or genetic means.

VII. Pharmaceutical Compositions

In some embodiments pharmaceutical compositions of the inventioncomprise a BibA polypeptide (with or without other active agents, asdisclosed above). In other embodiments pharmaceutical compositionscomprise a nucleic acid molecule encoding the BibA polypeptide (with orwithout nucleic acid molecules encoding other active agents, asdescribed above). Nucleic acid vaccines are described, for example, inRobinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly etal. (1997) Ann. Rev Immunol 15:617-648; Scott-Taylor & Dalgleish (2000)Expert Opin Investig Drugs 9:471-480; Apostolopoulos & Plebanski (2000)Curr Opin Mol Ther 2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-120;Dubensky et al. (2000) Mol Med 6:723-732; Robinson & Pertmer (2000) AdvVirus Res 55:1-74; Donnelly et al. (2000) Am J Respir Crit Care Med162(4 Pt 2):S190-193 Davis (1999) Mt. Sinai J. Med. 66:84-90. Typicallythe nucleic acid molecule is a DNA molecule, e.g., in the form of aplasmid. In other embodiments pharmaceutical compositions compriseantibodies which specifically bind to a BibA polypeptide

Immunogenic compositions of the invention are preferably vaccinecompositions. The pH of such compositions preferably is between 6 and 8,preferably about 7. The pH can be maintained by the use of a buffer. Thecomposition can be sterile and/or pyrogen-free. The composition can beisotonic with respect to humans. Vaccines according to the invention maybe used either prophylactically or therapeutically, but will typicallybe prophylactic and can be used to treat animals (including companionand laboratory mammals), particularly humans.

A. Pharmaceutically Acceptable Carriers

Compositions of the invention will typically, in addition to thecomponents mentioned above, comprise one or more “pharmaceuticallyacceptable carriers.” These include any carrier which does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers typically are large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. A composition mayalso contain a diluent, such as water, saline, glycerol, etc.Additionally, an auxiliary substance, such as a wetting or emulsifyingagent, pH buffering substance, and the like, may be present. A thoroughdiscussion of pharmaceutically acceptable components is available inGennaro (2000) Remington: The Science and Practice of Pharmacy 20th ed.,ISBN:0683306472.

B. Immunoregulatory Agents

1. Adjuvants

Vaccines of the invention may be administered in conjunction with otherimmunoregulatory agents. In particular, compositions will usuallyinclude an adjuvant. Adjuvants for use with the invention include, butare not limited to, one or more of the following set forth below:

a. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminum salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulfates, etc. (e.g. see chapters 8 & 9 of Vaccine Design . . . (1995)eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being preferred.The mineral containing compositions may also be formulated as a particleof metal salt (WO00/23105).

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

In one embodiment the aluminum based adjuvant for use in the presentinvention is alum (aluminum potassium sulfate (AlK(SO₄)₂)), or an alumderivative, such as that formed in-situ by mixing an antigen inphosphate buffer with alum, followed by titration and precipitation witha base such as ammonium hydroxide or sodium hydroxide.

Another aluminum-based adjuvant for use in vaccine formulations of thepresent invention is aluminum hydroxide adjuvant (Al(OH)₃) orcrystalline aluminum oxyhydroxide (AlOOH), which is an excellentadsorbant, having a surface area of approximately 500 m²/g.Alternatively, aluminum phosphate adjuvant (AlPO₄) or aluminumhydroxyphosphate, which contains phosphate groups in place of some orall of the hydroxyl groups of aluminum hydroxide adjuvant is provided.Preferred aluminum phosphate adjuvants provided herein are amorphous andsoluble in acidic, basic and neutral media.

In another embodiment the adjuvant of the invention comprises bothaluminum phosphate and aluminum hydroxide. In a more particularembodiment thereof, the adjuvant has a greater amount of aluminumphosphate than aluminum hydroxide, such as a ratio of 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1 or greater than 9:1, by weight aluminumphosphate to aluminum hydroxide. More particular still, aluminum saltsin the vaccine are present at 0.4 to 1.0 mg per vaccine dose, or 0.4 to0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6mg per vaccine dose.

Generally, the preferred aluminum-based adjuvant(s), or ratio ofmultiple aluminum-based adjuvants, such as aluminum phosphate toaluminum hydroxide is selected by optimization of electrostaticattraction between molecules such that the antigen carries an oppositecharge as the adjuvant at the desired pH. For example, aluminumphosphate adjuvant (isoelectric point=4) adsorbs lysozyme, but notalbumin at pH 7.4. Should albumin be the target, aluminum hydroxideadjuvant would be selected (iep 11.4). Alternatively, pretreatment ofaluminum hydroxide with phosphate lowers its isoelectric point, makingit a preferred adjuvant for more basic antigens.

b. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% TWEEN™80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer). See WO90/14837. See also, Podda, Vaccine (2001) 19:2673-2680; Frey et al., Vaccine (2003) 21:4234-4237. MF59 is used as theadjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

Particularly preferred adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Preferred submicron oil-in-wateremulsions for use herein are squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN™ 80□(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% SPAN 85™(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphosphoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MF59” (International Publication No. WO90/14837; U.S. Pat. Nos.6,299,884 and 6,451,325, and Ott et al., in Vaccine Design The Subunitand Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) PlenumPress, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene(e.g. 4.3%), 0.25-0.5% w/v TWEEN™ 80, and 0.5% w/v SPAN 85™ andoptionally contains various amounts of MTP-PE, formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.). For example, MTP-PE may be present in anamount of about 0-500 μg/dose, more preferably 0-250 μg/dose and mostpreferably, 0-100 μg/dose. As used herein, the term “MF59-0” refers tothe above submicron oil-in-water emulsion lacking MTP-PE, while the termMF59-MTP denotes a formulation that contains MTP-PE. For instance,“MF59-100” contains 100 μg MTP-PE per dose, and so on. MF69, anothersubmicron oil-in-water emulsion for use herein, contains 4.3% w/vsqualene, 0.25% w/v TWEEN™ 80, and 0.75% w/v SPAN 85™ and optionallyMTP-PE. Yet another submicron oil-in-water emulsion is MF75, also knownas SAF, containing 10% squalene, 0.4% TWEEN™ 80, 5% pluronic-blockedpolymer L121, and thr-MDP, also microfluidized into a submicronemulsion. MF75-MTP denotes an MF75 formulation that includes MTP, suchas from 100-400 μg MTP-PE per dose.

Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in WO90/14837 and U.S. Pat. Nos.6,299,884 and 6,451,325.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

c. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponins can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using High Performance ThinLayer Chromatography (HP-TLC) and Reversed Phase High Performance LiquidChromatography (RP-HPLC). Specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. Preferably, the saponin is QS21. A method of productionof QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulationsmay also comprise a sterol, such as cholesterol (see WO96/33739).

Combinations of saponins and cholesterols can be used to form uniqueparticles called Immunostimulating Complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of Quil A, QHA and QHC.ISCOMs are further described in EP0109942, WO96/11711 and WO96/33739.Optionally, the ISCOMS may be devoid of (an) additional detergent(s).See WO00/07621.

A review of the development of saponin based adjuvants can be found inBarr, et al., Advanced Drug Delivery Reviews (1998) 32:247-271. See alsoSjolander, et al., Advanced Drug Delivery Reviews (1998) 32:321-338.

d. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Q13-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin WO03/024480, WO03/024481, and Niikura et al., Virology (2002)293:273-280; Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto,et al., Journal of Infectious Diseases (2003) 188:327-338; and Gerber etal., Journal of Virology (2001) 75(10):4752-4760. Virosomes arediscussed further in, for example, Gluck et al., Vaccine (2002)20:B10-B16. Immunopotentiating reconstituted influenza virosomes (IRIV)are used as the subunit antigen delivery system in the intranasaltrivalent INFLEXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl5:B17-23} and the INFLUVAC PLUS™ product.

e. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as:

i. Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipidA with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such“small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC 529. See Johnson et al.(1999) Bioorg Med Chem Lett 9:2273-2278.

ii. Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in Meraldi et al.,Vaccine (2003) 21:2485-2491; and Pajak, et al., Vaccine (2003)21:836-842.

f. Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine may be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,Nucleic Acids Research (2003) 31(9): 2393-2400; WO02/26757 andWO99/62923 for examples of possible analog substitutions. The adjuvanteffect of CpG oligonucleotides is further discussed in Krieg, NatureMedicine (2003) 9(7): 831-835; McCluskie, et al., FEMS Immunology andMedical Microbiology (2002) 32:179-185; WO98/40100; U.S. Pat. No.6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003)31 (part 3): 654-658. The CpG sequence may be specific for inducing aTh1 immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs arediscussed in Blackwell, et al., J. Immunol. (2003) 170(8):4061-4068;Krieg, TRENDS in Immunology (2002) 23(2): 64-65 and WO01/95935.Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla, et al., BBRC (2003) 306:948-953; Kandimalla, etal., Biochemical Society Transactions (2003) 31 (part 3):664-658; Bhagatet al., BBRC (2003) 300:853-861 and WO03/035836.

g. ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (i.e., E. coli heat labile enterotoxin “LT),cholera (“CT”), or pertussis (“PT”). The use of detoxifiedADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211and as parenteral adjuvants in WO98/42375. Preferably, the adjuvant is adetoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use ofADP-ribosylating toxins and detoxified derivatives thereof, particularlyLT-K63 and LT-R72, as adjuvants can be found in the followingreferences: Beignon, et al., Infection and Immunity (2002)70(6):3012-3019; Pizza, et al., Vaccine (2001) 19:2534-2541; Pizza, etal., Int. J. Med. Microbiol (2000) 290(4-5):455-461; Scharton-Kersten etal., Infection and Immunity (2000) 68(9):5306-5313; Ryan et al.,Infection and Immunity (1999) 67(12):6270-6280; Partidos et al.,Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., Vaccines (2003)2(2):285-293; and Pine et al., (2002) J. Control Release (2002)85(1-3):263-270. Numerical reference for amino acid substitutions ispreferably based on the alignments of the A and B subunits ofADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol(1995) 15(6):1165-1167.

h. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres (Singh et al. (2001) J. Cont. Role. 70:267-276) ormucoadhesives such as cross-linked derivatives of polyacrylic acid,polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Chitosan and derivatives thereof may also beused as adjuvants in the invention. See WO99/27960.

i. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable and nontoxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide co glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

j. Liposomes

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0626 169.

k. Polyoxyethylene ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. WO99/52549. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers orester surfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group:polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steorylether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

l. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al.,“Preparation of hydrogel microspheres by coacervation of aqueouspolyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payneet al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug.Delivery Review (1998) 31(3):185-196.

m. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and Nacetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

n. Imidazoquinoline Compounds

Examples of imidazoquinoline compounds suitable for use adjuvants in theinvention include Imiquimod and its analogues, described further inStanley, Clin Exp Dermatol (2002) 27(7):571-577; Jones, Curr OpinInvestig Drugs (2003) 4(2):214-218; and U.S. Pat. Nos. 4,689,338,5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916,5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612.

o. Thiosemicarbazone Compounds

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in WO04/60308.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

p. Tryptanthrin Compounds

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in WO04/64759. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention:

-   -   (1) a saponin and an oil-in-water emulsion (WO99/11241);    -   (2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.        3dMPL) (see WO94/00153);    -   (3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.        3dMPL)+a cholesterol;    -   (4) a saponin (e.g., QS21)+3dMPL+IL 12 (optionally+a sterol)        (WO98/57659);    -   (5) combinations of 3dMPL with, for example, QS21 and/or        oil-in-water emulsions (See European patent applications        0835318, 0735898 and 0761231);    -   (6) SAF, containing 10% Squalane, 0.4% Tween 80, 5%        pluronic-block polymer L121, and thr-MDP, either microfluidized        into a submicron emulsion or vortexed to generate a larger        particle size emulsion.    -   (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2%        Squalene, 0.2% Tween 80, and one or more bacterial cell wall        components from the group consisting of monophosphorylipid A        (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),        preferably MPL+CWS (Detox™); and    -   (8) one or more mineral salts (such as an aluminum salt)+a        non-toxic derivative of LPS (such as 3dPML).

(9) one or more mineral salts (such as an aluminum salt)+animmunostimulatory oligonucleotide (such as a nucleotide sequenceincluding a CpG motif).

q. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophagecolony stimulating factor, and tumor necrosis factor.

Aluminum salts and MF59 are preferred adjuvants for use with injectableinfluenza vaccines. Bacterial toxins and bioadhesives are preferredadjuvants for use with mucosally-delivered vaccines, such as nasalvaccines.

The contents of all of the above cited patents, patent applications andjournal articles are incorporated by reference as if set forth fullyherein.

VIII. Therapeutic Methods

The invention provides methods for inducing or increasing an immuneresponse to S. agalactiae using the compositions described above. Theimmune response is preferably protective and can include antibodiesand/or cell-mediated immunity (including systemic and mucosal immunity).Immune responses include booster responses. Compositions comprisingantibodies can be used to treat S. agalactiae infections.

Diseases caused by GBS which can be prevented or treated according tothe invention include, but are not limited to, sepsis, meningitis innewborns, and newborn pneumonia. The compositions may also be effectiveagainst other streptococcal bacteria, e.g., GBS.

A. Tests to Determine the Efficacy of the Immune Response

One way of assessing efficacy of therapeutic treatment involvesmonitoring GBS infection after administration of the composition of theinvention. One way of assessing efficacy of prophylactic treatmentinvolves monitoring immune responses against the GBS antigens in thecompositions of the invention after administration of the composition.

Another way of assessing the immunogenicity of the component proteins ofthe immunogenic compositions of the present invention is to express theproteins recombinantly and to screen patient sera or mucosal secretionsby immunoblot. A positive reaction between the protein and the patientserum indicates that the patient has previously mounted an immuneresponse to the protein in question; i.e., the protein is an immunogen.This method may also be used to identify immunodominant proteins and/orepitopes.

Another way of checking efficacy of therapeutic treatment involvesmonitoring GBS infection after administration of the compositions of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses both systemically (such asmonitoring the level of IgG1 and IgG2a production) and mucosally (suchas monitoring the level of IgA production) against the GBS antigens inthe compositions of the invention after administration of thecomposition. Typically, GBS serum specific antibody responses aredetermined post-immunization but pre-challenge whereas mucosalGBS-specific antibody body responses are determined post-immunizationand post-challenge.

The vaccine compositions of the present invention can be evaluated in invitro and in vivo animal models prior to host, e.g., human,administration. A particularly useful mouse model is the Active MaternalImmunization assay described in Example 21, below. This is an in vivoprotection assay in which female mice are immunized with the testantigen composition. The female mice are then bred and their pups arechallenged with a lethal dose of GBS. Serum titers of the female miceduring the immunization schedule are measured as well as the survivaltime of the pups after challenge.

For example, groups of 4 CD-1 outbred female mice 6-8 weeks old (CharlesRiver Laboratories, Calco Italy) are immunized with one or more GBSantigens (e.g., 20 μg of a BibA polypeptide suspended in 100 μl of PBS).Each group receives 3 doses at days 0, 21 and 35. Immunization isperformed through intra-peritoneal injection of the protein with anequal volume of Complete Freund's Adjuvant (CFA) for the first dose andIncomplete Freund's Adjuvant (IFA) for the following two doses. In eachimmunization scheme negative and positive control groups are used.Immune response is monitored by using serum samples taken on day 0 and49. The sera are analyzed as pools from each group of mice.

The immune response may be one or both of a TH1 immune response and aTH2 response. The immune response may be an improved or an enhanced oran altered immune response. The immune response may be one or both of asystemic and a mucosal immune response. Preferably the immune responseis an enhanced system and/or mucosal response.

An enhanced systemic and/or mucosal immunity is reflected in an enhancedTH1 and/or TH2 immune response. Preferably, the enhanced immune responseincludes an increase in the production of IgG1 and/or IgG2a and/or IgA.

Preferably the mucosal immune response is a TH2 immune response.Preferably, the mucosal immune response includes an increase in theproduction of IgA.

Activated TH2 cells enhance antibody production and are therefore ofvalue in responding to extracellular infections. Activated TH2 cells maysecrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immuneresponse may result in the production of IgG1, IgE, IgA and memory Bcells for future protection.

A TH2 immune response may include one or more of an increase in one ormore of the cytokines associated with a TH2 immune response (such asIL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1,IgE, IgA and memory B cells. Preferably, the enhanced TH2 immuneresponse will include an increase in IgG1 production.

A TH1 immune response may include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a TH1 immuneresponse (such as IL-2, IFNγ, and TNFβ), an increase in activatedmacrophages, an increase in NK activity, or an increase in theproduction of IgG2a. Preferably, the enhanced TH1 immune response willinclude an increase in IgG2a production.

Immunogenic compositions of the invention, in particular, immunogeniccomposition comprising a BibA polypeptide of the present invention (ornucleic acid molecule encoding a BibA polypeptide) may be used eitheralone or in combination with other GBS antigens optionally with animmunoregulatory agent capable of eliciting a Th1 and/or Th2 response.

The invention also comprises an immunogenic composition comprising oneor more immunoregulatory agent, such as a mineral salt, such as analuminium salt and an oligonucleotide containing a CpG motif. Mostpreferably, the immunogenic composition includes both an aluminium saltand an oligonucleotide containing a CpG motif. Alternatively, theimmunogenic composition includes an ADP ribosylating toxin, such as adetoxified ADP ribosylating toxin and an oligonucleotide containing aCpG motif. Preferably, one or more of the immunoregulatory agentsinclude an adjuvant. The adjuvant may be selected from one or more ofthe group consisting of a TH1 adjuvant and TH2 adjuvant, furtherdiscussed below.

The compositions of the invention will preferably elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address a GBS infection. This immune response willpreferably induce long lasting (e.g., neutralizing) antibodies and acell mediated immunity that can quickly respond upon exposure to a BibApolypeptide.

In addition to a BibA polypeptide (or nucleic acid molecule encoding aBibA polypeptide), an immunogenic composition can comprise one or moreGBS antigen(s) which elicits a neutralizing antibody response and one ormore GBS antigen(s) which elicit a cell mediated immune response. Inthis way, the neutralizing antibody response prevents or inhibits aninitial GBS infection while the cell-mediated immune response capable ofeliciting an enhanced Th1 cellular response prevents further spreadingof the GBS infection. Preferably, the immunogenic composition comprisesone or more GBS surface antigens and one or more GBS cytoplasmicantigens, such as a cytoplasmic antigen capable of eliciting a Th1cellular response.

B. Preparation of Compositions

The compositions of the invention may be prepared in various forms. Forexample, a composition can be prepared as an injectable, either as aliquid solution or a suspension. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g., a lyophilized composition). A composition can beprepared for oral administration, such as a tablet or capsule, as aspray, or as a syrup (optionally flavored). A composition can beprepared for pulmonary administration, e.g., as an inhaler, using a finepowder or a spray. A composition can be prepared as a suppository orpessary. A composition can be prepared for nasal, aural or ocularadministration e.g., as drops. A composition can be in kit form,designed such that a combined composition is reconstituted just prior toadministration to a patient. Such kits may comprise one or more GBS orother antigens in liquid form and one or more lyophilized antigens.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of a BibA polypeptide (or a nucleic acid moleculeencoding a BibA polypeptide) or BibA antibodies, as well as any othercomponents, as needed, such as antibiotics. An “immunologicallyeffective amount” is an amount which, when administered to anindividual, either in a single dose or as part of a series, increases ameasurable immune response or prevents or reduces a clinical symptom.

In another embodiment, the antibiotic is administered subsequent to theadministration of a composition of the invention or the compositioncomprising the one or more surface-exposed and/or surface-associated GBSantigens of the invention. Examples of antibiotics suitable for use inthe treatment of a GBS infection include but are not limited topenicillin or a derivative thereof.

C. Methods of Administration

Compositions of the invention will generally be administered directly toa patient. The compositions of the present invention may beadministered, either alone or as part of a composition, via a variety ofdifferent routes. Certain routes may be favored for certaincompositions, as resulting in the generation of a more effective immuneresponse, preferably a CMI response, or as being less likely to induceside effects, or as being easier for administration.

Delivery methods include parenteral injection (e.g., subcutaneous,intraperitoneal, intravenous, intramuscular, or interstitial injection)and rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal(e.g., see WO 99/27961), transcutaneous (e.g., see WO02/074244 andWO02/064162), intranasal (e.g., see WO03/028760), ocular, aural, andpulmonary or other mucosal administration.

By way of example, the compositions of the present invention may beadministered via a systemic route or a mucosal route or a transdermalroute or it may be administered directly into a specific tissue. As usedherein, the term “systemic administration” includes but is not limitedto any parenteral routes of administration. In particular, parenteraladministration includes but is not limited to subcutaneous,intraperitoneal, intravenous, intraarterial, intramuscular, orintrasternal injection, intravenous, intraarterial, or kidney dialyticinfusion techniques. Preferably, the systemic, parenteral administrationis intramuscular injection. As used herein, the term “mucosaladministration” includes but is not limited to oral, intranasal,intravaginal, intrarectal, intratracheal, intestinal and ophthalmicadministration.

Teenagers and children, including toddlers and infants, can receive avaccine for prophylactic use; therapeutic vaccines typically areadministered to teenagers or adults. A vaccine intended for children mayalso be administered to adults e.g., to assess safety, dosage,immunogenicity, etc.

The immunogenic compositions of the present invention may beadministered in combination with an antibiotic treatment regime. In oneembodiment, the antibiotic is administered prior to administration of acomposition of the invention.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunization scheduleand/or in a booster immunization schedule. In a multiple dose schedulethe various doses may be given by the same or different routes e.g., aparenteral prime and mucosal boost, a mucosal prime and parenteralboost, etc.

The amount of active agent in a composition varies depending upon thehealth and physical condition of the individual to be treated, age, thetaxonomic group of individual to be treated (e.g., non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. The amount will fall in arelatively broad range which can be determined through routine trials.

IX. Kits

The invention also provides kits comprising one or more containers ofcompositions of the invention or their components. Compositions can bein liquid form or can be lyophilized, as can individual components ofthe compositions. Suitable containers for the compositions include, forexample, bottles, vials, syringes, and test tubes. Containers can beformed from a variety of materials, including glass or plastic. Acontainer may have a sterile access port (for example, the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle).

The kit can further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other buffers, diluents,filters, needles, and syringes. The kit can also comprise a second orthird container with another active agent, for example an antibiotic.

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity against S. agalactiae. Thepackage insert can be an unapproved draft package insert or can be apackage insert approved by the Food and Drug Administration (FDA) orother regulatory body.

X. Screening Methods

The invention provides assays for screening test compounds that bind toor modulate the activity of BibA. A test compound preferably (1) bindsto the coiled-coil domain and blocks the interaction of BibA withcomplement, e.g., C4 binding protein, or the formation of BibA dimers;or (b) binds to the proline-rich domain and blocks the binding of BibAto host epithelial cells; (c) binds to the N-terminal domain of BibA andblocks the binding of BibA to IgA; or (d) binds to various portions ofBibA and blocks the binding of IgG to the protein. Assays can be carriedout using full-length BibA protein or BibA polypeptides of theinvention.

A. Test Compounds

Test compounds can be pharmacologic agents already known in the art orcan be compounds previously unknown to have any pharmacologicalactivity. The compounds can be naturally occurring or designed in thelaboratory. They can be isolated from microorganisms, animals, orplants, can be produced recombinantly, synthesized by chemical methodsknown in the art, or obtained using any of the numerous combinatoriallibrary methods known in the art, including but not limited to,biological libraries, spatially addressable parallel solid phase orsolution phase libraries, synthetic library methods requiringdeconvolution, the “one-bead one-compound” library method, and syntheticlibrary methods using affinity chromatography selection.

B. Assays

Any method known in the art can be used to detect binding between a testcompound and a domain of BibA or disruption of binding between a domainof BibA and its biological target.

In some binding assays, either the test compound or the BibA protein orpolypeptide can comprise a detectable label, such as a fluorescent,radioisotopic, chemiluminescent, or enzymatic label, such as horseradishperoxidase, alkaline phosphatase, or luciferase. Methods of detectingsuch labels are well known in the art. Alternatively, binding can bedetermined without labeling either of the interactants. See, e.g.,McConnell et al., Science 257, 1906-12, 1992. Technologies such asreal-time Bimolecular Interaction Analysis (BIA) (Sjolander &Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al., Curr.Opin. Struct. Biol. 5, 699-705, 1995) also can be used.

In other embodiments, a BibA protein or polypeptide can be used as a“bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., Cell 72, 223-232, 1993; Maduraet al., J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al.,BioTechniques 14, 920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696,1993; and Brent WO94/10300), to identify other proteins which bind to orinteract with various domains of BibA.

Assays such as those described in the Examples below can be used todetect whether binding between various domains of BibA and thebiological targets of those domains is disrupted or prevented by a testcompound.

All patents, patent applications, and references cited in thisdisclosure are expressly incorporated herein by reference in theirentireties. The above disclosure generally describes the presentinvention. A more complete understanding can be obtained by reference tothe following specific examples, which are provided for purposes ofillustration only and are not intended to limit the scope of theinvention.

Example 1 BibA can Form Dimers

This example demonstrates that BibA can form dimers. In ahigh-resolution fractionation, molecules elute from the matrix pores inorder of decreasing size. Smaller molecules have greater access to thepores of the matrix and hence move down the column. See FIGS. 3A and 3B.

Example 2 BibA Protein Surface-Association

GBS bacteria were incubated with secondary FITC-conjugated α-mouse IgGantibody alone. Bacteria were also treated with mouse serum immunizedwith Freund adjuvant (α-PBS) as a negative control. FIGS. 4A and 4Billustrate the negative control on plots labeled “α-PBS.” BibA levelsare expressed as a change in mean of fluorescence between cells treatedwith α-BibA serum and pre-immune serum. Bacteria incubated withpre-immune serum were compared to bacteria treated with α-BibA serum toobtain a change in mean of fluorescence (Δ mean).

BibA protein of strains 2603, 18RS21 and H36B exhibitedsurface-association but strains 515 and CJB111 did not (Δmean=0). Seealso FIG. 5.

Example 3 BibA Protein Clusters

GBS strain 515 (pAM401-SAG2063) was grown overnight in THB medium (10ml). Bacterial cells from 1 ml of the overnight culture were resuspendedin 5 ml of fresh THB medium and grown at 37° C. up to OD 0.5 (stationaryphase). Bacteria were then centrifuged for 10 minutes at 3000 rpm atroom temperature, washed, and resuspended in 1 ml of PBS.Formvar-carbon-coated nickel grids were floated on drops of GBSsuspensions for 10 minutes. The grids were then fixed in 2% PFA for 15minutes and placed in blocking solution (PBS containing 1% normal rabbitserum and 1% BSA). The grids were then floated on drops of primaryantiserum against BibA (mαBibA) diluted 1:50 in blocking solution for 30minutes at room temperature, washed with 6 drops of blocking solution,and floated on secondary antibody conjugated to 10 nm gold particlesdiluted 1:25 in 1% BSA for 30 minutes. The grids were then washed with 4drops of PBS and then with 4 drops of distilled water and air dried. Thegrids were examined using a GEOL 1200 EX 11 transmission electronmicroscope.

FIG. 6 shows the presence of BibA protein clusters.

Example 4 BibA Expression on the Surface of Strain 515 pAM401

BibA gene (SAG2063) including its own promoter and terminator was clonedinto pAM401 vector using BamHI and SalI restriction sites as illustratedin FIG. 7A. GBS strains 2603 V/R. and 515 Ia were transformed with thisconstruct.

FACS analysis showed that BibA protein is exposed on the 515(pAM401-SAG2063) surface at high levels. FIG. 7B.

Example 5 Increased Expression of BibA on the Surface of Strain 2603

FACS analysis showed that the exposure of BibA protein on the 2603(pAM401-SAG2063) surface is increased respect the 2603 wt strain. Theresults are shown in FIG. 8.

Example 6 Secreted form of BibA

GBS protein extracts were separated via SDS-PAGE and transferred to anitrocellulose membrane. Proteins were then overlaid with a mouse α-BibApolyclonal antibody and stained with HRP-conjugated secondary antibody.As FIG. 10 illustrates, in the separation of BibA strains 515 andCJB111, the BibA protein was found only in the culture supernatant (thesecreted protein fraction). This demonstrates that the truncated form ofthe BibA protein in strains 515 and CJB111, which lack the proline-richmotif, is expressed in the secreted. See FIG. 10.

Example 7 BibA Binds C4 Binding Protein (C4BP)

Recombinant BibA proteins were dried onto a nitrocellulose membrane andincubated with recombinant c4-binding protein. Bound protein was thendetected using a mouse a-C4BP monoclonal antibody and stained withHRP-conjugated antibody. FIG. 11A is a dot blot in which BibA protein atdifferent concentrations is stained with the HRP-conjugated antibody,demonstrating that BibA binds C4 binding protein.

Recombinant BibA protein was separated by SDS-PAGE, blotted onto anitrocellulose membrane and then incubated with recombinant C4-bindingprotein. Bound protein was then detected using a mouse a-C4BP monoclonalantibody and stained with HRP-conjugated antibody. FIG. 11B shows aWestern blot that confirms that BibA binds C4 binding protein.

Example 8 BibA Protein Binds to the Surface of Epithelial Cells

ME180 cervical cells were incubated both in the presence and in theabsence of BibA protein followed by the addition of a mouse α-BibApolyclonal antibody. The ME180 cervical cells were then stained withFITC-conjugated α-mouse IgG secondary antibody. A positive control wasobtained by treating the ME180 cervical cells only with theFITC-conjugated α-mouse IgG secondary antibody (i.e., in the absence ofmouse α-BibA polyclonal antibody). The analysis was repeated with Caco2intestinal cells, A549 alveolar cells, and 16HBE140 bronchial cells. Asnegative control we used GBS7 protein, which was cloned identically toBibA.

BibA binding, expressed as Dmean channel values, was measured by FACScancytometer as difference in fluorescence intensity between cell incubatedwith or without BibA. Results are shown in FIG. 12. The “secondaryantibody only” area indicates cells treated with FITC-conjugatedantibody alone. BibA binding, expressed as Dmean channel values, wasmeasured by FACScan cytometer as difference in fluorescence intensitybetween cell incubated with or without BibA. As negative control we usedGBS7 protein, which was cloned identically to BibA.

Example 9 BibA Binds to the Surface of Epithelial Cells by theProline-Rich Motif

Epithelial cells were incubated in the presence of biotinylated BibAprotein or biotinylated BibA fragments and then stained withFITC-conjugated streptavidin. The purple area indicates cells treatedwith FITC-conjugated streptavidin alone. BibA binding, expressed asDelta mean channel values, was measured by a FACScan cytometer as thedifference in fluorescence intensity between cells incubated with orwithout proteins.

The results are shown in FIG. 13. These findings demonstrate that BibAbinds to the surface of epithelial cells by the proline-rich motif. Seealso FIGS. 25A and 25B; FIG. 26.

Example 10 Purified Human-IgG Binds to BibA Protein

Purified GBS3-His, GBS3-Nt-His (Nt), GBS3-Nt1-His (Nt1), GBS3-T-His (T),GBS3-Ct-His (Ct), GBS M protein (M1) (positive control) and GBS104(negative control) were separated on SDS-4%-12% PAGE gel (200V) andtransferred to nitrocellulose membrane (35V, 1 hr, 15 min). Thenitrocellulose membrane was blocked for 1 hr at RT with 5% Milk-PBS-0.1%Tween20 (PBS-T) and overlaid with immunoglobulins (human-IgA orhuman-IgG) in PBS-T for 1 hr at RT. The membrane was washed three timeswith PBS-T, overlaid with secondary HRP conjugated antibodies (1:1000)in 5% Milk-PBS-0.1% Tween20, and washed three times with PBS-T. Positivebinding to immunoglobulins was detected using an ECL™ substrate.

The results are shown in FIG. 14. The results demonstrate that thefull-length protein and fragments of the protein bind to human IgG withdifferent affinities.

Example 11 BibA-His is Specific for Human and Rabbit IgG

BibA-His was separated on SDS-PAGE and then transferred to anitrocellulose membrane. After blocking, the membranes were incubatedwith serum of different species and then probed with anti-IgG antibodyconjugated with HRP. The proteins were revealed with a colorimetric kit.

The results are shown in FIG. 17 and Table 1. These results demonstratethat BibA-His is specific for human and rabbit IgG and does not bindmouse IgG.

Example 12 Purified Human-IgA Binds to BibA Protein

Purified BibA-His, BibA-Nt-His (Nt), BibA-Nt1-His (Nt1), BibA-T-His (T),BibA-Ct-His (Ct), GBS M protein (M1) (positive control) and GBS104(negative control) were separated on SDS-4%-12% PAGE gel (200V) andtransferred to nitrocellulose membrane (35V, 1 hr, 15 min). Thenitrocellulose membrane was blocked for 1 hr at RT with 5% Milk-PBS-0.1%Tween20 (PBS-T) and overlaid with human-IgA-HRP conjugated in PBS-T for1 hr at RT. The membrane was washed three times with PBS-T. Positivebinding to immunoglobulins was detected using an ECL™ substrate. Theprotein was blocked by incubating the membrane with 5% milk-PBS-T for 1hr at RT. The membrane was incubated with human IgG-HRP (5 μg/ml) inPBS-T for 1.5 hr, then washed three times with PBS-T. Positive bindingto immunoglobulins was detected using an ECL™ substrate (PIERCE kit:SuperSignal West Pico Chemiluminescent Substrate) and 4-CN kit(BIO-RAD).

Native BibA protein and portions of BibA were examined via dot blot.

The results are shown in FIG. 15 and Table 2. These results demonstratethat the N-terminal portion of BibA binds to purified human IgA.

BibA-His and BibA fragments were digested with trypsin for 15 minutesand separated on SDS-PAGE gel. An overlay with h-IgA-HRP (1 ml/ml) wasperformed on the proteins transferring on nitrocellulose membrane. Theresults are shown in FIG. 16. Tryptic digestion produces a fragment of32 kDa which still binds h-IgA.

Human-IgA-FITC binding to the surface of 2603-BibA overexpressing mutantstrain is shown in FIG. 9. FACS analysis revealed an increment of IgAbinding to the surface of BibA overexpressing mutants.

Example 13 Production of Complete and Truncated Forms of BibA

Plasmids encoding complete or truncated form of BibA proteins wereconstructed as follows. Domains of BibA were amplified by PCR using 2603genome as the template. The oligonucleotide primers used are listed inthe Table 1. Forward primers all contained NdeI restriction sites. Thereverse primers all contained XhoI restriction sites. PCR product weredigested with NdeI and XhoI, gel purified and ligated with NdeI and XhoIrestricted pET21b(+). All constructs were verified by DNA sequencing.The recombinant proteins were expressed His-tag proteins.

GBS3-His is the entire form of BibA (GBS3)GBS3-Nt-His is the coiled-coil domain of BibAGBS3-Nt1-His is the coiled-coil domain without the first 180 aaGBS3-Ct-His is the proline-rich domain of BibAGBS3-Nt3-His contained the proline-rich domain and a portion ofcoiled-coil domain GBS3-T-His contained the first 180 aa GBS3-Hisfrom 34aa to 609aa (SEQ ID NO: 20) Forward NdeI 5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ (SEQ ID NO: 21) Reverse XhoI 5′-CCCGCTCGAG

-3′ Nucleotide sequence (SEQ ID NO: 22) CACGCGGATACTAGTTCAGGAATATCGGCTTCAATTCCTCATAAGAAACAAGTTAATTTAGGGGCGGTTACTCTGAAGAATTTGATTTCTAAATATCGTGGTAATGACAAAGCTATTGCTATACTTTTAAGTAGAGTAAATGATTTTAATAGAGCATCACAGGATACACTTCCACAATTAATTAATAGTACTGAAGCAGAAATTAGAAATATTTTATATCAAGGACAAATTGGTAAGCAAAATAAACCAAGTGTAACTACACATGCTAAAGTTAGTGATCAAGAACTAGGTAAGCAGTCAAGACGTTCTCAAGATATCATTAAGTCATTAGGTTTCCTTTCATCAGACCAAAAAGATATTTTAGTTAAATCTATTAGCTCTTCAAAAGATTCGCAACTTATTCTTAAATTTGTAACTCAAGCCACGCAACTGAATAATGCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGCAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGCAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCAGAGGTACTAAAGAAGATAAAAAACCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGAAGCTAAGCCAGAAGCTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAACCAGAGGCTAAGCCAGAAGCTAAACCAGAGGCTAAGTCAGAAGCTAAACCAGAGGCTAAGCTAGAAGCTAAACCAGAGGCCAAACCAGCAACCAAAAAATCGGTTAATACTAGCGGAAACTTGGCGGCTAAAAAAGCTATTGAAAACAAAAAGTATAGTAAAAAATTACCATCAACGGGTGAAGCC

Amino acid sequence of the fragment (SEQ ID NO: 23)MetHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKKLPSTGEAASPLLAIVSLIVMLSAGLITLEHHHHHH GBS3-Nt-His from 34aa to 394aa (SEQ ID NO: 24)Forward NdeI 5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ (SEQ ID NO: 25)Reverse XhoI 5′-CCCGCTCGAGACCTCTGGTAAGGTCTTGAA-3′Nucleotide sequence (SEQ ID NO: 26):CACGCGGATACTAGTTCAGGAATATCGGCTTCAATTCCTCATAAGAAACAAGTTAATTTAGGGGCGGTTACTCTGAAGAATTTGATTTCTAAATATCGTGGTAATGACAAAGCTATTGCTATACTTTTAAGTAGAGTAAATGATTTTAATAGAGCATCACAGGATACACTTCCACAATTAATTAATAGTACTGAAGCAGAAATTAGAAATATTTTATATCAAGGACAAATTGGTAAGCAAAATAAACCAAGTGTAACTACACATGCTAAAGTTAGTGATCAAGAACTAGGTAAGCAGTCAAGACGTTCTCAAGATATCATTAAGTCATTAGGTTTCCTTTCATCAGACCAAAAAGATATTTTAGTTAAATCTATTAGCTCTTCAAAAGATTCGCAACTTATTCTTAAATTTGTAACTCAAGCCACGCAACTGAATAATGCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGCAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGCAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCAGAGGTAmino acid sequence of the fragment (SEQ ID NO: 27)MetHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDFNRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRRSQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTEGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGLEHHHHHH BibA-Nt1-Hisfrom 180aa to 394aa (SEQ ID NO: 28)Forward NdeI 5′-GGAATTCCATATGGCTGAATCAACAAAAGCTA-3′ SEQ ID NO: 29)Reverse XhoI 5′-CCCGCTCGAGACCTCTGGTAAGGTCTTGAA-3′Nucleotide sequence (SEQ ID NO: 30):GCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGGAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGGAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCA GAGGTAmino acid sequence of the fragment (SEQ ID NO: 31)MetAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGLEHHHHHH GBS3-Ct-His from 389aa to 622aa(SEQ ID NO: 32) Forward NdeI 5′-GGAATTCCATATGCCAGACCTTACCAGAGGT-3′(SEQ ID NO: 33) Reverse XhoI 5′-CCCGCTCGAGCGTAATAAGACCTGCACTT-3′Nucleotide sequence (SEQ ID NO: 34):CAAGACCTTACCAGAGGTACTAAAGAAGATAAAAAACCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGAAGCTAAGCCAGAAGCTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAACCAGAGGCTAAGCCAGAAGCTAAACCAGAGGCTAAGTCAGAAGCTAAACCAGAGGCTAAGCTAGAAGCTAAACCAGAGGCCAAACCAGCAACCAAAAAATCGGTTAATACTAGCGGAAACTTGGCGGCTAAAAAAGCTATTGAAAACAAAAAGTATAGTAAAAAATTACCATCAACGGGTGAAGCCGCAAGTCCACTCTTAGCAATTGTATCACTAATTGTTATGTTAAGTGCAGGTCTTATTACGAmino acid sequence of the fragment (SEQ ID NO: 35)MetQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKKLPSTGEAASPLLAIVSLIVMLSAGLITLEHHHHHH GBS3-Nt3-HisFrom 180aa to 622aa (SEQ ID NO: 36)Primer 7 NdeI 5′-GGAATTCCATATGGCTGAATCAACAAAAGCTA-3′ (SEQ ID NO: 37)Primer 9 XhoI 5′-CCCGCTCGAGCGTAATAAGACCTGCACTT-3′Nucleotide sequence (SEQ ID NO: 38):GCTGAATCAACAAAAGCTAAGCAAATGGCTCAAAATGACGTGGCCTTAATAAAAAATATAAGCCCCGAAGTCTTAGAAGAATATAAAGAAAAAATTCAAAGAGCTAGCACTAAGAGTCAAGTTGATGAGTTTGTAGGAGAAGCTAAAAAAGTTGTTAATTCCAATAAAGAAACGTTGGTAAATCAGGCCAATGGTAAAAAGCAAGAAATTGCTAAGTTAGAAAATTTATCTAACGATGAAATGTTGAGATATAATACTGCAATTGATAATGTAGTGAAACAGTATAATGAAGGTAAGCTCAATATTACTGCTGCAATGAATGCTTTAAATAGTATTAAGCAAGCAGCACAGGAAGTTGCCCAGAAAAACTTACAAAAGCAGTATGCTAAAAAAATTGAAAGAATAAGTTCAAAAGGATTAGCGTTATCTAAAAAGGCTAAAGAAATTTATGAAAAGCATAAAAGTATTTTGCCTACACCTGGATATTATGCAGACTCTGTGGGAACTTATTTGAATAGGTTTAGAGATAAACAAACTTTCGGAAATAGGAGTGTTTGGACTGGTCAAAGTGGACTTGATGAAGCAAAAAAAATGCTTGATGAAGTCAAAAAGCTTTTAAAAGAACTTCAAGACCTTACCAGAGGTACTAAAGAAGATAAAAAACCAGACGTTAAGCCAGAAGCCAAACCAGAGGCCAAACCAGACGTTAAGCCAGAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAGAAGCTAAGCCAGACGTTAAACCAAAGGCCAAACCAGACGTTAAGCCAGAAGCTAAGCCAGACGTTAAACCAGACGTTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAGGATAAGCCAGACGTTAAACCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGAAGCTAAGCCAGAAGCTAAGCCAGAGGCCAAACCAGAAGCTAAGCCAGACGTTAAGCCAGAAGCTAAACCAGACGTTAAACCAGAGGCTAAGCCAGAAGCTAAACCAGAGGCTAAGTCAGAAGCTAAACCAGAGGCTAAGCTAGAAGCTAAACCAGAGGCCAAACCAGCAACCAAAAAATCGGTTAATACTAGOGGAAACTTGGCGGCTAAAAAAGCTATTGAAAACAAAAAGTATAGTAAAAAATTACCATCAACGGGTGAAGCCGCAAGTCCACTCTTAGCAATTGTATCACTAATTGTTATGTTAAGTGCAGGTCTTATTACGAmino acid sequence of the fragment (SEQ ID NO: 39):MetAESTKAKQMAQNDVALIKNISPEVLEEYKEKIQRASTKSQVDEFVAEAKKVVNSNKETLVNQANGKKQEIAKLENLSNDEMLRYNTAIDNVVKQYNEGKLNITAAMNALNSIKQAAQEVAQKNLQKQYAKKIERISSKGLALSKKAKEIYEKHKSILPTPGYYADSVGTYLNRFRDKQTFGNRSVWTGQSGLDEAKKMLDEVKKLLKELQDLTRGTKEDKKPDVKPEAKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPEAKPDVKPKAKPDVKPEAKPDVKPDVKPDVKPEAKPEDKPDVKPDVKPEAKPDVKPEAKPEAKPEAKPEAKPEAKPEAKPDVKPEAKPDVKPEAKPEAKPEAKSEAKPEAKLEAKPEAKPATKKSVNTSGNLAAKKAIENKKYSKKLPSTGEAASPLLAIVSLIVMLSAGLITLE HHHHHHGBS3-T-His (SEQ ID NO: 40)Primer 1 NdeI 5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ (SEQ ID NO: 41)GBS3-T-Rev XhoI 5′-CCCGCTCGAGATTATTCAGTTGCGTGGCTTGAGT-3′Nucleotide sequence (SEQ ID NO: 42):CACGCGGATACTAGTTCAGGAATATCGGCTTCAATTCCTCATAAGAAACAAGTTAATTTAGGGGCGGTTACTCTGAAGAATTTGATTTCTAAATATCGTGGTAATGACAAAGCTATTGCTATACTTTTAAGTAGAGTAAATGATTTTAATAGAGCATCACAGGATACACTTCCACAATTAATTAATAGTACTGAAGCAGAAATTAGAAATATTTTATATCAAGGACAAATTGGTAAGCAAAATAAACCAAGTGTAACTACACATGCTAAAGTTAGTGATCAAGAACTAGGTAAGCAGTCAAGACGTTCTCAAGATATCATTAAGTCATTAGGTTTCCTTTCATCAGACCAAAAAGATATTTTAGTTAAATCTATTAGCTCTTCAAAAGATTCGCAACTTATTCTTAAATTTGTA ACTCAAGCCACGCAACTGAATAATAmino acid sequence of the fragment (SEQ ID NO: 43):MetHADTSSGISASIPHKKQVNLGAVTLKNLISKYRGNDKAIAILLSRVNDENRASQDTLPQLINSTEAEIRNILYQGQIGKQNKPSVTTHAKVSDQELGKQSRESQDIIKSLGFLSSDQKDILVKSISSSKDSQLILKFVTQATQLNNLEHHHHHH

Example 14 Experimental Procedures

Cell Culture

The human cervical epithelial cell line ME180 was purchased from theAmerican Type Culture Collection (ATCC, Rockville, Md.). ME180 cellswere maintained in RPMI 1640 medium with 10% heat-inactivated fetalbovine serum (FBS). The lung carcinoma cell line A549 (type II alveolarepithelial cells) and the colon carcinoma epithelial cell line Caco2also were supplied by the ATCC and were grown in DMEM supplemented with10% FBS, 4.5 g/L glucose and non-essential amino acids. The humanbronchial epithelial cell line 16HBE14, which is transformed with SV40large T antigen (Grifantini et al., 2002), was cultured in DMEMsupplemented with 10% FBS, 1.5 mM glutamine, and 100 μg/ml kanamycinsulfate.

Bacterial Strains and Growth Conditions

S. agalactiae strains 2603 V/R and 515 Ia were used in this study. Todetermine BibA protein conservation, we analyzed a panel of S.agalactiae strains. E. coli DH5α and DH10BT1 were used for cloningpurposes and E. coli BL21 (DE3) for expression of BibA fusion protein.S. agalactiae was cultivated at 37° C. in Todd-Hewitt broth (THB) up toOD600 0.4. S. agalactiae strains carrying the plasmid pAM401bibA weregrown in the presence of cloramphenicol (10 μg/ml). E. coli was grown inLuria broth. E. coli clones carrying the plasmids pAM401bibA,pJRS233ΔbibA or pET21(b)+ derivatives were grown in the presence ofcloramphenicol (20 μg/ml), erythromycin (400 μg/ml) or ampicillin (100μg/ml), respectively.

Construction of 2603 V/R bibA Deletion Mutant

The bibA gene was deleted in S. agalactiae strain 2603 V/R according tothe procedure described in Lauer et al., 2005. The in-frame deletionfragment was obtained by Splicing Overlap Extension (SOE) PCR using theprimers 5′-CCCG

ACTAGTGACAAACCTTGGAAT-3′ (SEQ ID NO:44),5′-GTCAGCACGGTTTGCCATAAACCGAAAGGTCTATCC-3′ (SEQ ID NO:45),5′-ACCTTTCGGTTTATGGCAAACGCTGCTGACATTG-3′ (SEQ ID NO:46) and 5′-CCCG

ACAGATAAGCCTAAGCGACTT-3′ (SEQ ID NO:47). The XhoI restriction enzymecleavage sites were incorporated at the 5′-end of the primer (bold anditalicized) to clone the fragment into the XhoI-digested pJRS233plasmid. After cloning the in frame deletion fragment in the pJRS233,the plasmid pJRS233ΔbibA was obtained.

The plasmid pJRS233ΔbibA was then transformed into the 2603 V/R strainby electroporation and transformants were selected after growth at 30°C. on agar plates containing 1 μg/ml erythromycin. Transformants werethen grown at 37° C. with erythromycin selection as described in Maguinet al., 1996. Integrant strains were serially passaged for 5 days inliquid medium at 30° C. without erythromycin selection to facilitate theexcision of plasmid pJRS233ΔbibA, resulting in the bibA deletion on thechromosome. Dilutions of the serially passaged cultures were plated ontoagar plates, and single colonies were tested for erythromycinsensitivity to confirm the excision of pJRS233ΔbibA.

Plasmid-Mediated Expression of BibA in S. agalactiae

The bibA gene including its own promoter and terminator was amplified byPCR from chromosomal DNA of S. agalactiae 2603 V/R using primers5′-CCCCGCC

CCAACCCTTATCAAAAGA-3′ (SEQ ID NO:48) and 5′-CTCTGCATG

CATAGAAACAACCCAAACCC-3′ (SEQ ID NO:49). The restriction enzymes cleavagesites BamHI and SalI were incorporated at the 5′-ends of the primers(bold and italicized) to clone the PCR product into the BamHI/SalIdigested E. coli-Streptococcus pAM401 expression construct. The plasmidpAM401bibA was obtained by cloning the bibA gene into pAM401. PlasmidpAM401bibA was transformed by electroporation into 2603 V/R and 515 Iastrains with subsequent cloramphenicol selection.

BibA Recombinant Protein Expression and Purification

In order to express the recombinant form of BibA, the open reading frameof the bibA gene from S. agalactiae 2603 was used as a template. Theconstruct was amplified by PCR using specific primers, which introducedNdeI and XhoI restriction enzyme sites:

(SEQ ID NO: 50) 5′-GGAATTCCATATGCACGCG GATACTAGTTCAGGA-3′ and(SEQ ID NO: 51) 5′-CCCGCTCGAGAATTGCTAAGAGTGG ACTTGC-3′.

In the case of BibA N-terminal construct (aa 34-394) the followingamplification primers were used:

(SEQ ID NO: 52) 5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ and(SEQ ID NO: 53) 5′-CCC GCTCGAGACCTCTGGTAAGGTCTTGAA-3′.

For BibA C-terminal construct (aa 389-622) the following primers wereused:

(SEQ ID NO: 54) 5′-GGAATTCCATATGCCAGACCTTACCAGAGGT-3′ and(SEQ ID NO: 55) 5′-CCCGCTCGAGCGTAATAAGACC TGCACTT-3′.

The PCR products were cloned into the pET21(b)+ vector, which was usedto transform E. coli BL21 (DE3) cells. BL21 (DE3) cells were grown inLB-Amp (100 μg/ml ampicillin) and induced with IPTG at finalconcentration of 1 mM for 3 hours. The resulting biomass was suspendedin 0.3M NaCl, 50 mM Na—PO4 buffer, pH 8.0, and cells were lysed by twopassages at 18,000 psi using a Basic Z Model Cell Disrupter (ConstantSystems Ltd., Daventry, UK). The sample was then loaded onto a His-TrapNi-Activated Chelating Sepharose FF column (Amersham Biosciences, Milan,Italy) at a flow rate of 5 ml/min. Bound proteins were then eluted fromthe column by running a gradient from 0 to 50% of 500 mM Imidazole, 0.3M NaCl, 50 mM Na phosphate buffer, pH 8.0 in 12 CV. The IMAC(Immobilized Metal Affinity Column) eluted material was collected in2.5-ml fractions, and the fractions containing the BibA-His protein werepooled. The collected pools were then loaded onto a HiLoad 26/60Superdex 200 gel filtration column (Amersham Biosciences, Milan, Italy).The protein was eluted isocratically at 2.5 ml/min flow rate collecting2.5-ml fractions.

Bacterial Extracts

GBS protein extracts were prepared by growing bacteria up to OD₆₀₀ 0.4.The resulting pellet washed in PBS and incubated for 1 hr at 37° C. in500 μl of Tris-HCl 50 mM (pH6.8) containing protease inhibitors and 400U/ml of mutanolysin (SIGMA, MO, USA). The bacterial suspension was thenpelleted and supernatants containing peptidoglycan-associated proteinswere used for Western blotting analysis of BibA. In order to prepare GBSextracts containing the secreted protein fraction, supernatant ofbacteria cultures grown to OD₆₀₀ 0.4 were collected and directly used inPAGE.

Fluorescence-Activated Cell Sorter Analysis

In order to quantify the exposure of BibA on bacterial surface, GBS wasgrown up to OD600 0.4 and incubated with rabbit anti-BibA serum orrabbit anti-PBS serum (negative control) in 0.1% BSA plus 20% of normalcalf serum (NCS) for 1 hr at 4° C. Bacteria were then washed in PBScontaining 0.1% BSA and incubated with phicoerytrin (PE)-conjugatedsecondary antibodies (Jackson Immuno Research Inc., PA, USA) for 45 minat 4° C. After washing, bacteria were fixed with 2% PFA for 20 min atRT, resuspended in 200 μl of PBS, and analyzed by a FACSscan flowcytometer (Becton Dickinson) using the Cell Quest software program fromBecton Dickinson.

In the binding assay, ME180 or A549 cells were mixed with differentconcentrations of BibA and incubated for 1 hr at 4° C. Cells weresubsequently incubated for 45 min. at 4° C. with rabbit anti-BibA serumin 5% FCS. Cells were then washed twice in PBS and incubated for 45 min.at 4° C. with the PE-conjugated secondary antibodies. Cell-boundfluorescence was analyzed with a FACS using the Cell Quest program. MFIvalues of cells incubated with or without protein were compared.

Association Assay

ME180 and A549 epithelial cells were infected with approximately 10bacteria/cell in infection medium (basal medium without antibiotics)supplemented with 2% FBS in 200 μl volumes. At the end of a 3-hourincubation at 37° C. in 5% CO2 (v/v), total colony-forming units(c.f.u.) were estimated after addition of 1% saponin to the wellscontents. Adhesiveness was quantified by determining the ratio ofcell-associated c.f.u. versus total c.f.u. present in the assay.

Immunogold Labeling and Electron Microscopy

GBS strains 2603 V/R, 2603ΔbibA, 515 Ia and 515pAM401bibA were grownovernight in THB medium (10 ml). Bacterial cells from 1 ml of theovernight culture were resuspended in 5 ml of fresh THB medium and grownat 37° C. up to OD 0.3 (exponential phase). Bacteria were thencentrifuged for 10 min at 3000 rpm (RT), washed and resuspended in 1 mlof PBS. Formvar-carbon-coated nickel grids were floated on drops of GBSsuspensions for 5 min. The grids were then fixed in 2% PFA for 5 min,and placed in blocking solution (PBS containing 1% normal rabbit serumand 1% BSA) for 30 min. The grids were then floated on drops of primaryantiserum against the BibA protein diluted 1:20 in blocking solution for30 min at RT, washed with six drops of blocking solution, and floated onsecondary antibody conjugated to 10-nm gold particles diluted 1:10 in 1%BSA for 30 min. The grids were examined by using a TEM GEOL 1200EX IItransmission electron microscope.

Confocal Immunofluorescence Microscopy

A549 cells were grown to confluence in a Lab-TekII Chamber Slide System(Nalgene) in 1 ml of DMEM supplemented with 10% FBS, 4.5 g/L glucose andnon-essential amino acids. Cells were then infected with bacteria at aMOI 10:1 and incubated at 37° C. for 2 hr. Cells were then fixed in 2%paraformaldehyde for 30 min at room temperature (RT) or at 4° C.overnight. After fixing, the monolayers were blocked with 3% BSA andincubated for 1 hr at RT with a mix of mouse anti-capsule and rabbitanti-BibA polyclonal antibodies diluted in 1% BSA. Bacteria were thenstained, for 1 hr at RT, with goat anti-mouse and anti-rabbit Alexafluor (Molecular Probes) conjugated antibodies (excitation at 568 nm and488 nm, respectively). F-actin was stained with Alexa Fluor 622conjugated phalloidin. The chamber walls were then removed from theglass slide. A Slow Fade reagent kit (Molecular Probes) used to mountcover slips. The slides were viewed with a Bio-Rad confocal scanningmicroscope.

Dot Blot and Western Blot Analyses

In dot blot analysis, purified recombinant BibA protein (range ˜2μg-0.01 μg) was absorbed to a nitrocellulose membrane by using a BIORADdot blot system. After saturation with 5% milk, the membrane wasincubated with 0.5 μg/ml of serum purified human-IgA (Pierce) or humanIgG (SIGMA). After washing, the membrane was incubated withHRP-conjugated rabbit anti-human IgA (Dako) or HRP-conjugated goatanti-human IgG (BD) and positive binding detected by ECL.

The same protocol was used to test the binding of BibA to C4BP. PurifiedC4BP derived from citrated human plasma was purchased from Kordia LifeScience, (Leiden, N. Dak.). Mouse monoclonal anti-C4BP antibodies(BIOTREND Chemikalien GmbH, Köln) were used to reveal C4BP binding toBibA.

Western blot analysis of BibA binding to Ig or C4BP was performed bytransferring SDS-PAGE separated proteins to nitrocellulose membranes(Portran). Membranes were then blocked in 5% milk and overlaid for 1 hrwith 5 μg/ml of (a) purified IgG from normal human serum (SIGMA); (b)purified IgG from normal mouse serum (SIGMA); c) purified IgG frombovine serum (SIGMA); d) purified IgA from human serum (Pierce); (e)purified IgA from human colostrums (SIGMA); or (f) human plasma C4BP(Kordia Life Science, ND). After washing, membranes were incubated withthe respective HRP-conjugated secondary antibodies, and detection wasperformed by ECL.

Sequence Analysis

The alignment of 2603 V/R (GenBank Accession Number NP_(—)689049; SEQ IDNO:56), 18RS21 (AAJO00000000; SEQ ID NO:57), 515 Ia (AAJP00000000; SEQID NO:58), NEM316 (NP_(—)736451; SEQ ID NO:59), H36B (AAJS00000000; SEQID NO:60), CJB111 (AAJQ00000000; SEQ ID NO:61), A909 (YP_(—)330593; SEQID NO:62; see also SEQ ID NO:67) and COH1 (AAJR00000000; SEQ ID NO:63)strains was performed using ClustalW (Thompson et al., 1994).

Example 15 Additional Evidence that BibA is Exposed on GBS Surface

As shown in FIG. 19A, FACS analysis of 2603 V/R strain grown atexponential phase (OD₆₀₀=0.35) revealed a shift in bacterialfluorescence after staining with anti-BibA antibody. This indicated thatBibA is exposed on GBS surface. This finding was further confirmed bytransmission immuno-electron microscopy (IEM) showing positiveimmunogold labeling of BibA on 2603 V/R surface (FIG. 19B). Western blotanalysis of 2603 V/R bacterial extracts showed the presence of a singleband recognized by anti-BibA antibodies in both peptidoglycan-associatedprotein fraction and bacteria supernatants (FIG. 19C). The bandidentified as BibA has an apparent MW of ≈80 kD (FIG. 19C), compared tothe expected MW of 66 kD. We believe that the presence of a prolin-richmotif in the C-terminal region of BibA is responsible for such adiscrepancy. Indeed, it is known that prolin-rich regions may retardprotein electrophoretic migration (Hollingshead et al., 1986).Comparative analysis of bacteria grown at exponential or stationaryphases revealed no differences in the expression of BibA as surfaceexposed or secreted protein (data not shown). As expected, BibA knockoutmutant strain showed nor BibA FAGS positive fluorescence neitherimmunogold surface labeling (FIGS. 19D and 19E).

In order to demonstrate that the anchoring of BibA to the cell wall wasdue to the presence of the LPXTG (SEQ ID NO:3) motif, we investigatedBibA surface exposure in the strain 515 Ia, in which, due to aframeshift, the protein is lacking the LPXTG (SEQ ID NO:3) motif andtherefore is predicted to be expressed in a truncated form (Tettelin etal., 2005). Both FACS analysis and IEM confirmed that in such a strainBibA was not surface exposed (FIGS. 19E and 19F). Moreover, Western blotanalysis showed that BibA was found in 515 Ia bacterial supernatant, butnot in the peptidoglycan associated fraction (FIG. 19G). The apparentmolecular weight of 38 kD is in agreement with the predicted truncatedform. When we introduced in the strain 515 Ia a plasmid carrying the2603 V/R region containing the bibA gene and its regulatory elements(pAM401bibA), BibA was translocated and anchored on the bacterialsurface, as demonstrated by Western blotting, FACS and IEM analysis(FIGS. 19H, 19I and 19L).

Example 16 BibA Specifically Binds to Human Immunoglobulins

Because sequence analysis of BibA indicated some similarity withstreptococcal immunoglobulin-binding proteins, we performed Westernblotting analysis of recombinant BibA overlaid with purifiedserum-derived immunoglobulins (Ig). Experimental positive control wasthe M1 protein of GBS, which is an IgG and IgA binding protein(Cunningham, 2000). The recently reported GBS pilus component proteinGBS104 (Lauer et al., 2005) was used as unrelated negative control. Asshown in FIG. 20A, BibA specifically bound to purified human serum IgG,but not to mouse or bovine IgG. On the other hand, M1 protein reactedwith human, mouse and bovine IgG isoforms at similar levels.

BibA binding to purified human serum- or secretory colostrums-derivedIgA was also tested. As for the M1 protein, BibA positively recognizesboth serum-derived and secretory IgA (FIG. 20B). In order to demonstratethat the binding properties were not due to the gel denaturingconditions, we performed native dot-blot experiments. Recombinant BibAprotein was serially diluted on nitrocellulose membrane and probed with0.5 μg/ml purified human serum IgG and IgA. As shown in FIGS. 20C and20D, probing of native BibA with Ig confirmed the binding to human IgGand IgA. The reactivity of BibA for human IgA appeared to be strongerthan that for human IgG. Indeed, a positive binding to IgA was alreadyobserved at a concentration of BibA of 0.4 μg, while for IgG theconcentration of BibA necessary to the binding was of 1.0 μg (FIG. 20D).On the contrary, a strong binding to IgG was detected only up BibA (FIG.20C).

In order to elucidate the BibA binding region to Ig, we generated twoconstructs comprising the N-terminal portion of BibA (aa 34-394) or theC-terminal (aa 400-600). These two BibA constructs have been tested forbinding to human IgG and IgA in overlay immunoblotting assays. As shownin FIG. 20E, BibA binding to human IgG resided prevalently in theN-terminal region of the protein, although some binding was observedalso associated to the C-terminal portion. On the other hand, thebinding to human IgA was exclusively associated to the N-terminalportion of BibA (FIG. 20F).

Example 17 BibA Binds to Human Complement Regulator C4bp

Because both BibA and M proteins bind to human IgA, we asked if thepreviously described (Carlsson et al, 2003) ability of M proteins tobind C4b-binding protein (C4bp) was also carried by BibA. We tested BibAbinding activity by C4bp overlay blots in both denaturing andnon-denaturing conditions. As shown in FIG. 21, recombinant BibAseparated in SDS-gel electrophoresis (FIG. 21A) or spotted in the nativeform on nitrocellulose membrane (FIG. 21B) highly reacts with C4bpoverlaid at 5 μg/ml. M1 similarly bound to C4bp in both conditions,while a negative control protein (GBS201), randomly chosen from the 2603V/R genome, did not bind. Of interest, BibA did not show any binding forthe alternative complement pathway regulator Factor H. BibA N-terminaland C-terminal constructs were also tested for C4bp binding. As shown inFIG. 21C, overlay blots of SDS-PAGE separated BibA showed that theN-terminal region of the protein was sufficient to specifically bind toC4bp. No binding was observed by the C-terminal portion.

Example 18 BibA Recombinant Protein Binds to Epithelial Cells

In silico prediction of BibA propensity to form coiled-coil regionssuggested an adhesive phenotype. We initially tested the capacity of therecombinant BibA, as expressed in the 2603 V/R strain, to bind to ME180cervical epithelial cells. BibA binding was performed by incubatingcells with different concentrations of the recombinant protein for 1 hat 4° C. A rabbit polyclonal serum raised against recombinant BibA wasused as primary antibody and the binding detected byR-Phycoerythrin-conjugated secondary antibody. To determine antibodyunspecific binding, cells were incubated with primary polyclonalantibodies in the absence of the protein. After incubation of ME180cells with increased concentrations of BibA, we found that the bindingof BibA reached a plateau at a concentration of ≈5 μg/ml.

As shown in FIG. 22A, because the binding of BibA to ME180 cells couldbe saturated, the affinity of recombinant BibA for its putative receptorwas estimated by plotting the mean of fluorescence intensity of theBibA-receptor complex versus the free BibA concentration (FIG. 22B). TheKd value was then calculated as the BibA concentration that determinesthe saturation of 50% of the putative receptors present on cells andevaluated to be in the order of ≈4×10⁻⁸ M. We also tested binding ofrecombinant BibA to intestinal (Caco2), pulmonary (A549) and bronchial(16HBE) epithelial cell lines. Incubation of these cells with 10 μg/mlof recombinant BibA significantly increased the mean of fluorescence ofthe BibA-receptor complex (FIG. 22C), even if the intensity of the shiftvaried among the different cell types.

Example 19 BibA is Involved in GBS Adhesion to Epithelial Cells

In order to confirm that recombinant BibA adhesive properties wereassociated to a functional role during interaction with epithelialcells, we performed association assays comparing the 2603 V/R wild typestrain to the isogenic BibA knockout mutant strain, which does notexpress the protein on the bacterial surface (FIG. 19D). As shown inFIGS. 23A and B, the absence of BibA on the surface of 2603 V/R strainsignificantly reduced GBS capacity to associate to both ME180 and A549cells (p<0.05). Complementation of the mutation by inserting thepAM401bibA plasmid restored the adhesive phenotype (data not shown). Theimpaired capacity of the 2603ΔbibA strain to adhere to epithelial cellswas also evident in confocal imaging experiments. 2603 V/R wild typestrain and the isogenic BibA knockout mutant strain were stained withrabbit anti-BibA and mouse anti-serotype V polyclonal antibodies.

As shown in FIGS. 23C and 23D, the number of bacteria associated toepithelial cells found in a microscopy field (magnification 20×) wasreduced in the BibA mutant strain. These results were in total agreementwith those obtained by association assay. Transformation of the 2603 V/Rwild type strain with the pAM401bibA plasmid was used as a tool toincrease, BibA exposure on bacterial surface. FACS experiments confirmedthat the 2603 pAM401bibA strain showed a 30% increase in the number offluorescence intensity channels compared to the wild type strain.Association assays demonstrated that BibA overexpression wasfunctionally related to the capacity of GBS to adhere to epithelialcells. Indeed, compared to wild type strain 2603 pAM401bibA strainshowed an increased adherence to both ME180 and A549 cells (FIGS. 23Aand 23B).

As previously shown in FIG. 19, we were able to express in the 515 Iastrain, which does not expose BibA on the surface, the 2603 V/R form ofBibA (515 pAM401bibA). In order to demonstrate that such expression wasassociated to a functional adhesive phenotype as for 2603 V/R wild typestrain, we compared association levels to epithelial cells between 515Ia wild type strain and 515 pAM401bibA. We observed that BibA exposureon 515 Ia surface resulted in a significant increase in the percentageof associated bacteria to both ME180 and A549 cells (FIGS. 23A and 23B).This phenotype was also evident by confocal microscopy imaging (FIGS.23E and 23F).

Example 20 BibA Genomic Characterization

Genomic analysis on the recently sequenced genomes of 2603 V/R (Tettelinet al., 2002), NEM316 type III (Glaser et al., 2002), COH1 type III,CJB111 type V, 515 type Ia, 18RS21 type II and A909 type Ia (Tettelin etal., 2005) strains shows that bibA is present in all these strains,although interrupted by the insertion of two putative transposases onthe opposite strand in A909. This insertion causes the interruption ofthe reading frame at nucleotide 580. The bibA gene present on 515 Iastrain shows a frame-shift, which results in a truncated form of theprotein (FIG. 19G), consisting of the N-terminal 376 amino acids andlacking the proline-rich and cell wall anchoring regions. A similarframe-shift occurs in CJB111 strain, resulting in the translation of theN-terminal 469 amino acids. Such a protein fragment was found in CJB111supernatants. Western blot analysis of GBS supernatants from 31 strainsrepresenting the most common serotypes, revealed that BibA was presentin 81% of the strains (Table 4).

On the other hand, FACS analysis showed that BibA was expressed on thesurface of 58% of the strains, while in 19% of them BibA was exclusivelyrecovered in bacterial supernatants. However, BibA exposure on bacterialsurface totally correlated with the presence in the supernatant.

In general, two different types of sequence variability can be observedamong the different BibA proteins. The first is the presence of avariable number of brief amino acid modules, which can be observedeither within the N-terminal domain, or within the proline-rich tract.In particular, the presence of a region of 97 amino acids, holding therepeats IKAESIN (SEQ ID NO:65) and KIQXKXNT (SEQ ID NO:66) is observedwithin the N-terminal domain in A909, CJB11 and H36B, while the numberof copies of the PEAK/PDVK modules varies between 17 and 42 (see Table3). This suggests the insertion/excision of transposable elements.

The second source of sequence variation consists of a non-repetitivetract of 97 residues within proline-reach region, which characterizes515, NEM316, H36B, CJB111 and A909 strains. Collectively, the sequenceanalysis reveals that the protein exists in three different variants,one formed by strains 2603, 18RS21, and COH1, the other by NEM316 and515, and the last one originated by CJB111, H36B and A909 (FIG. 24).However, the multiple alignment of the BibA amino acid sequences, showsthat the protein is generally well conserved (amino acid identity rangesbetween 63.3 and 100% among N-terminal domains of different strains),with the exception of COH1, whose N-terminal region shows on averageabout 25% of amino acid identity to the other alleles.

Example 21 Active Maternal Immunization Assay

A maternal immunization/neonatal pup challenge model of GBS infection isused to verify the protective efficacy of the antigens in mice. SeeRodewald et al., J. Infect. Diseases 166, 635, 1992. CD-1 female mice(6-8 weeks old) are immunized before breeding. The mice receive 20 μg ofprotein per dose when immunized with a single antigen and 60 μg ofprotein per dose (15 μg of each antigen) when immunized with thecombination of antigens. Mice are bred 2-7 days after the lastimmunization. Within 48 h of birth, pups are injected intraperitoneallywith 50 μl of GBS culture. Challenge inocula are prepared starting fromfrozen cultures diluted to the appropriate concentration with THB beforeuse. In preliminary experiments, the challenge doses per pup for eachstrain tested is determined to cause 90% lethality. Survival of pups ismonitored for 2 days after challenge. Protection is calculated as(percentage deadControl minus percentage deadVaccine) divided bypercentage deadControl multiplied by 100. Data are evaluated forstatistical significance by Fisher's exact test.

Example 22 BibA Knock-Out Mutant Strain is Cleared More Easily in HumanBlood

The importance of BibA expression in bacterial survival in vivo wasassessed in freshly drawn blood from human donors. GBS was grown up toOD₆₀₀ 0.4, washed, and resuspended in PBS. Inocula of 10⁴ CFU in 100 μlwere mixed with 300 μl of freshly drawn human blood using heparin asanticoagulant. The tubes were incubated for 3 hours with agitation at37° C., and dilutions were plated for determination of CFU.

As shown in Table 5, 2603 V/R wild-type strain and the isogenic2603ΔbibA knock-out mutant strain were compared for the capacity toreplicate in whole human blood. The bacterial survival index wascalculated as the ratio between the number of bacteria recovered at theend of the assay versus bacteria at time 0. We tested five individualdonors and found that 2603 V/R wild-type strain proliferated in humanblood 5 times more efficiently than the 2603ΔbibA mutant strain.However, survival indexes varied among the different donors. In threedonors, where the wild-type strain replicated slowly in blood (from 5 to14 times), the bibA knock-out mutant strain was almost cleared. Bycontrast, in two donors where the wild-type strain replicated highly inblood (77 and 41 times), the bibA mutant strain still proliferated,although less efficiently (37 and 5 times, respectively). These findingssuggest that in donors with a reduced anti-bactericidal activity, thecontribution of BibA to GBS survival in blood is less pronounced.

Example 23 Complement-Mediated GBS Killing by PolymorphonuclearLeukocytes (PMNs) is Affected by BibA Expression

Phagocytic clearance of GBS by human blood is mainly mediated by PMNswhich kill opsonized bacteria in the presence of complement. The killingby human PMNs of the 2603 V/R wild-type strain and the bibA mutantstrains were compared. PMNs were obtained from heparin-anticoagulatedvenous blood of normal, healthy volunteers by dextran sedimentation,Ficoll-Hypaque density gradient centrifugation, and hypotonic lysis ofresidual erythrocytes, as described in Maione et al., Science 309,148-50.

To prepare bacteria for killing experiments, 2603 V/R and 2603ΔbibAgrown in THB medium were collected in mid-log pase of growth (OD₆₀₀0.4), washed in HBSS (Gibco-BRL), and adjusted to a density of 10⁷CFU/ml. GBS (10⁶ CFU) were opsonized for 15 minutes at 37° C. with 5%human serum containing anti-GBS antibodies. The bacteria were incubatedfor 3 h with PMNs and the number of surviving bacteria was determined byquantitative plating on TSA plates.

Incubation of GBS with human serum alone resulted in no killing. Asshown in FIG. 30, at the end of the 3 h incubation, the bibA mutantstrain was killed more efficiently than the wild-type strain. Indeed,only 7% of the mutant bacteria survived phagocytosis by PMNs, comparedto 30% for the wild-type strain. Heat inactivation of complementresulted in survival of both wild-type and bibA mutant strains, whichreplicated 5- to 6-fold during the time span of the experiment (FIG.30).

TABLE 1 Rab- bit h- h- Bo- Human Mouse se- Total IgG1 IgG2 hIgG3 vineserum serum rum h-IgG (λ) (κ) (κ) IgG IgG IgG IgG BibA- +++ − − − − +++− + His BibA- ++ − − − − ++ − − Nt-His BibA- + − − − − − − − Ct-His

TABLE 2 Human serum Human IgA colostrum-IgA BibA-His +++ +++ BibA-Nt-His++ + BibA-Ct-His − ++

TABLE 3 GBS strain Serotype Surface* Supernatant° MW (kDa) A909 Ia − − — 515 Ia − + 38 DK1 Ia +++ + 90 2177 Ib/c − + 90 5551 Ib/c +++ + 80 H36BIb ++ + 90 2129 Ib + + 80 7357b Ib ++ + 80 5518 Ib − − — 18RS21 II ++ +80 3050 II +++ + 80 5401 II − + 70 2141 II ++ + 80 M732 III − − — M781III − + 60 COH1 III − − — 5435 III/R − + 60 5376 III/R − − — 1998 III/R+++ + 80 2274 IV − + 40 1999 IV + + 90 2210 IV + + 80 2603 V +++ + 80CJB111 V − + 48 5364 V ++ + 80 2110 V/c ++ + 80 2189 VIII − − —JM9130013 VIII ++ + 98 JMU071 VIII ++ + 98 CJB110 NT + + 60 1169 NT ++ +60 *(−) MFI 0-50 (+) MFI 50-100 (++) MFI 100-200 (+++) MFI 200-300 °(−)WB negative (+) WB positive

TABLE 4 GBS strain PEAK/PDVK modules COH1 17 NEM316 21 A909 25 CJB111 29H36B 38 2603 42  515 42 18R821 42

TABLE 5 Survival Index % survival (% survival) Donor A Donor B Donor CDonor D Donor E (mean ± SD) 2603 V/R 5.2 (100) 77.5 (100)  5.4 (100)14.7 (100) 41.3 (100) 100 2603ΔbibA 0.7 (12.9) 37.6 (48.5) 0.76 (14.1) 1.3 (8.8)  5.4 (13.1) 19.5 ± 14.6

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1. A purified preparation of antibodies which specifically bind to a polypeptide, wherein a portion of the polypeptide consists of (1) a “coiled-coil domain” which is at least 95% identical to amino acids 34-394 of SEQ ID NO:1 (BibA); (2) a “proline-rich domain” which is at least 95% identical to amino acids 389-622 of SEQ ID NO:1; or (3) the coiled-coil domain of (1) and the proline-rich domain of (2); wherein the portion is free of other contiguous amino acid sequences of a protein with the amino acid sequence SEQ ID NO:1.
 2. The purified preparation of antibodies of claim 1, wherein the amino acid sequence of the proline-rich domain is amino acids 389-622 of SEQ ID NO:1.
 3. The purified preparation of antibodies of claim 1, wherein the amino acid sequence of the coiled-coil domain is amino acids 34-394 of SEQ ID NO:1.
 4. The purified preparation of antibodies of claim 1 wherein the portion consists of the coiled-coil domain.
 5. The purified preparation of antibodies of claim 1 wherein the portion consists of the proline-rich domain.
 6. The purified preparation of antibodies of claim 1 wherein the portion consists of the coiled-coil domain and the proline-rich domain.
 7. The purified preparation of antibodies of claim 6 wherein the amino acid sequence of the portion is at least 95% identical to SEQ ID NO:4.
 8. The purified preparation of antibodies of claim 6 wherein the amino acid sequence of the portion is SEQ ID NO:4.
 9. The purified preparation of antibodies of claim 1, wherein the antibodies are polyclonal.
 10. The purified preparation of antibodies of claim 1, wherein the antibodies are monoclonal.
 11. The purified preparation of antibodies of claim 1, wherein the antibodies are selected from the group consisting of F(ab′)₂ fragments, F(ab) fragments, F_(v) molecules, non-covalent heterodimers, single-chain F_(v) molecules, dimeric antibody fragment constructs, trimeric antibody fragment constructs, minibodies, diabodies, and chimeric antibodies.
 12. The purified preparation of antibodies of claim 1, wherein the antibodies are humanized.
 13. The purified preparation of antibodies of claim 1, wherein the antibodies are human antibodies.
 14. A composition comprising: the purified preparation of antibodies of claim 1; and a pharmaceutically acceptable carrier.
 15. The composition of claim 14, further comprising an antibody which specifically binds to a polypeptide antigen which is useful in a pediatric vaccine.
 16. The composition of claim 15, wherein the polypeptide antigen selected from the group consisting of N. meningitidis, S. pneumoniae, Bordetella pertussis, Moraxella catarrhalis, Clostridium tetani, Chorinebacterim diphteriae, respiratory syncytial virus, polio virus, measles virus, mumps virus, rubella virus, and rotavirus polypeptide antigens.
 17. The composition of claim 14, further comprising an antibody which specifically binds to a polypeptide antigen which is useful in a vaccine for elderly or immunocompromised individuals.
 18. The composition of claim 17, wherein the polypeptide antigen selected from the group consisting of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Legionella pneumophila, Listeria monocytogenes, influenza virus, and parainfluenza virus polypeptide antigens.
 19. A method of treating an S. agalactiae infection comprising administering to an individual in need thereof the composition of claim
 14. 